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Division of Physiology, Department of Medicine, University of California, La Jolla, California 92093
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ABSTRACT |
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 TOP
 ABSTRACT
 INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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Hypoxic exercise increases VEGF expression and the formation of new capillaries. In addition to hypoxia-inducible factor regulation at the transcriptional level, VEGF message stabilization is also a key regulatory step for VEGF expression. In vitro experiments have identified Hu protein R (HuR) as a potential posttranscriptional regulator of VEGF gene expression. Here, we report that in rat skeletal muscle (gastrocnemius muscle), 1) HuR binds to a known regulatory sequence located in the VEGF mRNA 3'-untranslated region (1,631-1,678 bp); 2) HuR specifically binds to the A/U-rich element AUUUUA (1,665-1,670 bp) and an additional A/U-rich region containing the consensus sequence UUUUUUA (1,658-1,664 bp); 3) binding of HuR to VEGF mRNA is seen already 5 min after acute ischemia, remaining elevated throughout a 60-min ischemic period; 4) a second inducible HuR-VEGF mRNA binding factor is evident 30 and 60 min postischemia; and 5) VEGF mRNA and protein levels are increased 20 and 30 min, respectively, after acute ischemia. These findings suggest that acute ischemia induces a rapid binding of HuR to the VEGF mRNA 3'-untranslated region. In skeletal muscle, this specific protein-RNA interaction may be an important posttranscriptional regulatory mechanism for increasing VEGF expression in response to hypoxia or acute ischemia.
angiogenesis; acute ischemia; hypoxia
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INTRODUCTION |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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REDUCED OXYGEN
TENSION has been suggested as a primary stimulus for VEGF
expression and angiogenesis after exercise (25). This
hypothesis is supported by numerous studies showing an augmented increase in VEGF levels after exercise in acute hypoxia (2, 4,
6). Hypoxia-inducible factor (HIF)-1 has also been shown to be
an important transcriptional regulator of VEGF expression (15). However, whereas we have previously reported that
VEGF mRNA levels were increased in the skeletal muscle of rats exposed to moderate hypoxia (rats breathing 12% O2), HIF-1
protein levels were not altered (34). These findings imply
that a HIF-1-independent mechanism for VEGF expression may also play
a role in the hypoxia-induced VEGF response in skeletal muscle.
Hu proteins, first identified as specific tumor antigens in paraneoplastic neuropathy or Hu syndrome (12), are the homologues of Drosophila protein embryonic lethal abnormal visual (ELAV). ELAV proteins belong to a family of RNA binding proteins necessary for neuronal differentiation (17). So far, four Hu proteins have been identified. Three of these proteins, HuB (Nel-N1), HuC, and HuD, are expressed in terminally differentiated neurons and neuroendocrine tumors (1, 10, 21), whereas only HuR (HuA) is expressed ubiquitously in all tissues (28). Hu proteins bind with a high affinity and specificity to adenylate/uridylate-rich elements (AREs) located in the 3'-untranslated region (3'-UTR) of several mRNAs. The binding interaction of HuR to AREs either stabilizes mRNA transcripts, enhances translation, or a combination of these posttranscriptional regulatory steps (11, 13, 14, 30). Many biological conditions, including hypoxia (26), heat shock (16), and exposure to short-wave ultraviolet light (18), specifically increase the binding of HuR to specific AREs located in the 3'-UTR of mRNA species. Interestingly, heat shock increases HuR functional activity, leading to an attenuation of mRNA degradation without altering the total amount of HuR present in HeLa cells (16). After identification of a shuttling sequence (HNS) located in the hinge region between the second and third RNA recognition motifs of HuR, it has been proposed that the translocation of HuR between the nucleus and cytoplasm is a critical step that allows HuR to bind to its RNA consensus sequence and inhibit mRNA degradation (5, 22).
Posttranscriptional factor binding to AREs is a common regulatory mechanism for several immediate-early and short-lived genes, including c-myc, c-fos, Id, N-myc, GM-CSF, and GAP 43 (7, 10, 23, 24, 27, 29, 33). VEGF mRNA also contains several classic AREs located in its 3'-UTR. These specific consensus sequences have the potential to bind Hu proteins and regulate VEGF expression at the posttranscriptional level (25). Studies of cells cultured in vitro have identified a major site for HuR binding and posttranscriptional regulation between 1,631 and 1,678 bp of the VEGF 3'-UTR (26).
In the present study, we show that HuR binding activity to two distinct AREs located within the 3' regulatory region of VEGF mRNA is induced by acute ischemia in rat gastrocnemius muscle. This HuR binding activity is followed by an increase in VEGF expression.
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METHODS |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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Animal experiments. Six groups, each containing six young adult female Wistar rats (8-12 wk), were used in this experiment. After rats were anesthetized with pentobarbital sodium (40-60 mg/kg ip), the gastrocnemius muscle was exposed, and the hind legs were tightly clamped with plastic cable ties to completely abolish blood flow. The gastrocnemius muscle was then removed at one of the following times after the onset of ischemia: 0, 5, 10, 20, 30, and 60 min. The muscle was then immediately frozen in liquid nitrogen. Six rats were used at each of the above time points, and rats at each time point constituted each of the six groups.
Synthesis of RNA probes.
To synthesize RNA probes for the EMSA, a series of truncated RNA
probes, which span regions of the rat VEGF mRNA 3'-UTR sequence (25), were synthesized from DNA oligonucleotides and
cloned into pBluscript II KS (Stratagene, CA) KpnI and
PstI sites (Fig. 1). A short
66-bp region of pBluescript II KS (transcription of T3 to
PstI site) served as the control probe. Plasmid constructs were digested with PstI, transcribed with T3 polymerase
(Stratagene) in the presence of [32P]UTP, and purified
with a RNase-free spin column (Roche). For competition assays, a
nonradioactive probe was synthesized from plasmid 59-71 using
nonradioactive UTP.
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Electromobility shift assay.
EMSA was modified from a protocol described previously
(35). Frozen samples of the gastrocnemius muscle were
homogenized in a buffer containing 100 mM NaCl, 50 mM Tris (pH 7.4),
0.5 mM Triton X-100, 1 mM DTT, 50 mM NaF, 0.5 mM NaVO3, and
an EDTA-free protease inhibitor cocktail tablet (Roche; Indianapolis,
IN). Homogenates were centrifuged at 14,000 rpm for 10 min at 4°C, and the supernatants were collected. Protein concentrations were measured with Bio-Rad protein assay kits (Hercules, CA). HuR-mRNA binding assays included the 200-µg muscle protein sample,
32P-labeled mRNA probe (
106 counts/min per
reaction), and 50 µl of reaction buffer [50 mM NaCl, 50 mM KCl, 1 mM
MgCl2, 50 mM Tris · HCl (pH 7.6), 0.5% NP-40, 50 mM NaF, 5 mM DTT, and 250 µg/ml tRNA]. The reaction mixtures were
incubated for 15 min at 25°C, followed by the addition of RNase T1 (2 µl, 25 U/ml), and incubated for an additional 30 min at 25°C. One
microliter of 2% bromophenol blue was added to each reaction mixture,
and the samples were immediately loaded onto a 6% native acrylamide
gel in 1× Tris-borate-EDTA buffer and electrophoresed at 250 V for
3 h. Gels were dried and exposed to BioMax film (Kodak; Rochester, NY) for autoradiography.
Binding assay for VEGF mRNA 3'-UTR. Because there was only minimal binding of HuR to the VEGF mRNA in control protein samples, protein isolated from muscles subjected to 20 min of ischemia was used to delineate the specific HuR binding regions in the VEGF 3'-UTR. The whole binding region (probe 31-78) was analyzed and compared with a series of truncated probes (probes 59-78, 59-71, 59-65, 66-71, 31-71, 31-65, and 31-58) to determine the presence of the muscle-specific factors. In mutation assays, the core binding region [59-71 (5'-UUUUUUAAUUUUA-3')] was used as a control and compared with the binding ability of different mutated probes [60Mt (5'-UCGCGUAAUUUUA-3'), 68Mt (5'-UUUUUUAAUCGUA-3'), and 60,68Mt (5'-UCGCGUAAUCGUA-3')].
Nonradioactive probe competition and HuR antibody shift assay. The core probe 59-71 and the same protein samples used in the deletion assays were also used in assays to determine the specificity of binding. In the nonradioactive probe competition assay, either 10- or 100-fold of nonradioactive probe was added along with the radioactive probe in the reaction mixture. In the supershift assay, 1 µl HuR (0.2 µg) antibody 3A2 (Santa Cruz Biotechnology) was included in the reaction mixture. The reaction mixtures were subsequently analyzed for HuR binding activity by EMSA as described above.
Northern blot analysis of VEGF mRNA. Northern blot analyses were performed as described previously (4). Briefly, the muscle was homogenized in guanidinium buffer, and total RNA was isolated with the phenol-chloroform method (9). Total RNA (30 µg) was size fractionated by electrophoresis on a 6.6% formaldehyde-1% agarose gel and transferred to a Zeta probe membrane (Bio-Rad). The membranes were subsequently hybridized with rat VEGF cDNA probes (5) labeled with [32P]dCTP using a random primer labeling kit (Stratagene). Blots were exposed to BioMax film (Kodak) and quantitated by densitometry. The same membranes were stripped and rehybridized with rat 18S cDNA probe labeled with [32P]dCTP to control for uniformity of loading in each lane of the gel.
Western blot analysis for HuR and VEGF. Western blot analysis was modified from the method of Gallouzi et al. (16). The same protein samples analyzed in EMSA were used for the Western blot assays. Skeletal muscle protein (50 µg/lane) was denatured in loading buffer [0.25 M Tris (pH 6.8), 20% glycerol, 4% SDS, and 0.05% bromophenol blue] by boiling for 5 min. Samples were electrophoresed on 12% SDS-PAGE and transferred to Immobilon P membranes (Millipore; Bedford, MA). Membranes were incubated with blocking buffer [5% dry milk, 0.02% (vol/vol) Tween 20, and 0.01% (vol/vol) anti-foam A (Sigma; St. Louis, MO) in PBS] for 2 h to block nonspecific protein binding. Anti-HuR 3A2 (Santa Cruz Biotechnology) (18) or anti-VEGF (Santa Cruz Biotechnology) antibodies were incubated with the blots, respectively, as primary antibodies. After incubation overnight at 4°C, membranes were washed with PBS-0.02% Tween 20 and incubated with horseradish peroxidase-conjugated rabbit anti-mouse IgG (Amersham; Cleveland, OH) secondary antibodies. The VEGF- and HuR-specific signals were detected by chemiluminescence using an ECL kit (Amersham) and exposed to BioMax film (Kodak).
Data processing and statistics. The bands from the Northern and Western blots and EMSA were quantitated by densitometric analysis from digital scanned images using HP DeskScan (Hewlett-Packard; Wilmington, DE) and Gel-Pro Analyzer software (Media Cybernetics). Statistical significance was determined by ANOVA, and P < 0.05 was considered significant.
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RESULTS |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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Identification of VEGF mRNA 3'-UTR protein binding activity in rat
skeletal muscle.
In Fig. 2, no binding activity was
detected using a nonspecific control probe. A clear band, reflecting
slower mobility binding activity, was detected using probe 31-78
and protein extracts isolated from 20-min ischemic
gastrocnemius muslce. Probe 31-78 alone (without protein sample)
showed no shifted bands. Binding activity indicates that factors
present in the gastrocnemius muscle bind to the core regulatory area
(1,631-1,678 bp) of the VEGF mRNA 3'-UTR (17).
Binding activity equivalent to that observed with probe 31-78 was
also observed with probe 59-71 (Fig. 2), and this was
competitively inhibited with a 10-fold excess of nonradioactive probe
59-71 (Fig. 3). Effective
competition of the band observed in samples taken after 20 min of
ischemia and the two bands observed in after 1 h of
ischemia were also competitively inhibited by a 100-fold excess
of nonradioactive probe (Fig. 3). These observations suggest that
binding activities are specific for the ARE-containing sequence.
Furthermore, HuR-specific antibody 3A2 added to the reaction mixture
containing protein extracts isolated from muscle exposed to 20 min of
ischemia resulted in a supershifted band that was observed in
addition to the constitutive binding activity. This supershifted band
confirms the presence of HuR in the constitutive binding activity.
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Delineation of specific HuR binding sites in the core regulatory
region of the VEGF mRNA 3'-UTR.
In our analysis, by using a series of truncated probes that span
regions of the VEGF 3'-UTR (Figs. 1 and 2), the maximum binding activity was detected from 1,659-1,671 bp (regions
1,631-1,658 and 1,672-1,678 both deleted). Binding
activity did not appear to be enhanced with longer RNA probes
59-78, 31-71, or 31-78. Furthermore, binding activity
was also detected with probe 59-65, which contains the UUUUUUA
consensus sequence (Fig. 2). No binding activity was detected when very
short RNA probe 66-71, containing the AUUUUA consensus sequence,
was used for mobility shift assays. To further identify the precise
nucleotides involved in these protein-RNA interactions, site-specific
RNA mutant probes were used in a gel mobility shift assay (Fig.
4). Mutation of one ARE sequence
downstream of probe 59-71 (5'-UUUUUUAAUUUUA-3') to mutated probe 68Mt (5'-UUUUUUAAUCGUA-3') or mutated probe 60Mt (5'-UCGCGUAAUUUUA-3') did not alter protein-RNA binding activity. However, mutated probe 60,68Mt
(5'-UCGCGUAAUCGUA-3'), which altered both
ARE consensus sequences, eliminated all protein-RNA binding activity.
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Increased HuR-VEGF mRNA binding activity in skeletal muscle exposed
to acute ischemia.
The constitutive HuR band progressively increased with the duration of
ischemia (Fig. 5A).
Increases above skeletal muscle protein values from
nonischemic, control rats were as follows: 1.9 ± 0.17-fold at 5 min, 3.7 ± 0.25-fold at 10 min, 4.2 ± 0.31-fold at 15 min, 8.5 ± 0.76-fold at 20 min, 13.0 ± 1.86-fold at 30 min, and 12.3 ± 2.11-fold at 60 min (Fig.
5B). Upon complete hindlimb ischemia for 30 min or
longer, an additional band with a slower electrophoretic mobility
showing binding activity was observed in the gel mobility shift assay
using the probe 59-71 (Fig. 5A). This
ischemia-induced binding activity was competed by 100-fold excess nonradioactive probe 59-71 (Fig. 3).
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Increased HuR-VEGF mRNA binding is temporally correlated with
ischemia-induced VEGF expression.
VEGF mRNA and protein levels in the ischemic gastrocnemius
muscle were evaluated by Northern and Western blots, respectively. Northern blots (Fig. 6A)
demonstrated that VEGF mRNA levels were increased 20 min after
ischemia (2.4 ± 0.19-fold) and were further increased
over the time of ischemia (2.9 ± 0.31-fold at 30 min and
3.6 ± 0.38-fold at 60 min; Fig. 6B). Western blots
revealed that HuR protein levels did not change throughout the
ischemic exposure (Fig. 7, A and
B), whereas VEGF protein
levels were significantly increased (Fig. 7, A and
B): 2.1 ± 0.2-fold at 30 min and 3.2 ± 0.40-fold
at 60 min after the onset of ischemia.
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DISCUSSION |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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HuR-VEGF 3'-UTR binding in rat gastrocnemius muscle in vivo. HuR is a 36-kDa protein with three RNA binding domains. One is suggested to bind with the poly(A) tail, whereas the other two have been implicated in ARE recognition (28, 29). Like its homologue ELAV in Drosophila, HuR has been reported in vitro to interact with many mRNA 3'-UTR regions, including those found in VEGF mRNA (7, 10, 16, 23, 24, 27, 28, 33). In the current experiment, we established that HuR is present in rat skeletal muscle (Figs. 6 and 7) and that its binding activity to the VEGF 3'-UTR could be evaluated by HuR-mRNA EMSA (Fig. 2). HuR binding activity is highly specific, and this was confirmed by competition experiments with an excess of nonradioactive probe containing the ARE sequences as well as the ability of HuR-specific antibody to supershift binding activity (Fig. 3). Our RNA EMSA results also indicate that a probe containing 13 bp of nucleotides, UUUUUUAAUUUUA (1,659-1,671 bp in the VEGF 3'-UTR), is the minimal sequence required to detect HuR binding activity in protein extracts from the intact hindlimb gastrocnemius muscle.
HuR binding sites in VEGF mRNA 3'-UTR. AREs containing several adenosine and uridine ribonucleotides in the mRNA 3'-UTR have been identified in several mRNA species. This is often true for many mRNA species with a short half-life (8). In vitro RNA binding experiments have also shown that these ARE-containing 3'-UTR are regulated by HuR binding, which increases the stability of these early expressed transcripts (7, 10, 16, 23, 24, 27, 28, 33). The standard ARE specific for Hu proteins is AUUUUA (8), and this exact sequence is present in the VEGF mRNA 3'-UTR between 1,666 to 1,671 bp. This site has been suggested as an active binding site for HuR based on in vitro experiments in 3T3 cells (26). In the present work, we show that this AUUUUA site is also important for HuR binding in skeletal muscle in vivo. Mutations of the AUUUUA consensus sequence to AUGCUA abolish HuR binding to this region (Fig. 4). Interestingly, our results also indicate that a minimal sequence of AUUUUA by itself (probe 66-71) is not sufficient to retain binding activity of muscle-specific transcription factors (Fig. 2). These results suggest that base pairs immediately adjacent to the AUUUUA sequence are necessary to retain binding. Surprisingly, HuR efficiently binds to a second sequence between base pairs 1,659 and 1,665 containing UUUUUUA (Fig. 2). Mutations to this sequence also abolish HuR binding activity (Fig. 4). These results indicate that the core regulatory region of VEGF in the mRNA 3'-UTR is from 1,659 to 1,671 bp. Within this region, HuR can bind to two ARE sites, and binding of HuR to each of these AREs appears to occur with the same affinity. The functional relationship between these two binding sites will require further investigation.
Ischemia increases HuR-VEGF mRNA 3'-UTR binding in rat gastrocnemius muscle. Previous research has shown that the binding activity of HuR-VEGF mRNA 3'-UTR is oxygen sensitive. Upon exposure to 1% oxygen, the VEGF mRNA half-life increases dramatically in 293T cells, which overexpress HuR (26). In the present study, we found that the constitutive binding of HuR to the sequence containing base pairs 1,659-1,671 increased as early as 5 min after ischemia and gradually increased further over the next 30 min (Fig. 5, A and B). In addition to the constitutive HuR binding activity, an inducible binding activity to the 1,659-1,671 bp sequence was detected at 30 and 60 min after the onset of ischemia (Fig. 5A). This ischemia-inducible band was specific and could be competed by an excess of nonradioactive probe 59-71. However, this inducible binding activity could not be shifted by incubation with HuR antibody 3A2 in EMSA (Fig. 3). The possible reasons for absence of a supershifted HuR band might be that 1) a chemical modification of HuR under hypoxia blocks the binding of antibody 3A2, 2) more protein domains are bound during extended hypoxia and mask the HuR antibody binding site, or 3) another Hu-like protein may also bind to core AREs within the VEGF mRNA 3'-UTR but is not recognized by the particular HuR-specific antibody used in our experiments.
Skeletal muscle VEGF and HuR responses to ischemia.
VEGF is a hypoxia-inducible growth factor (31). Its gene
expression can be regulated by HIF-1 at the transcriptional level and
by HuR at the posttranscriptional level (25). In our
previous study (34), we found that both HIF-1 and VEGF
levels in the rat gastrocnemius muscle increased similarly and
substantially during exercise, whereas only VEGF but not HIF-1
increased in the gastrocnemius muscle exposed to moderate hypoxia. This
implies that there is a sensitive-regulatory pathway for increasing
VEGF gene expression in response to hypoxia that does not involve the expression of HIF-1 or its transacting binding to the VEGF promoter. In
the present study, we found that the HuR binding activity specific for
the VEGF mRNA 3'-UTR was elevated as early as 5 min after the onset of
ischemia (Fig. 5), before the increase in VEGF mRNA and
protein. Increased VEGF expression was detected 20 and 30 min after
ischemic insult, respectively (Figs. 6 and 7). These results
suggest an alternative HIF-independent and immediate
posttranscriptional mechanism for increasing VEGF expression in
response to acute ischemia. The mechanism by which HuR
regulates mRNA levels has been proposed to involve HuR binding to hnRNA
in the nucleus and subsequent translocation to the cytoplasm. In the
cytoplasm, it is protected from degradation by nuclear RNase enzymes
and is thus available for translation. HuR itself returns to nucleus by
a HuR-specific import shuttling system. The translocation of HuR to the
cytoplasm will enhance its protective function by attenuating mRNA
degradation rates (5, 22). A similar mechanism has been observed in HeLa cells. Heat shock dramatically increases HuR binding
to poly (A) RNA located in the cytoplasm of HeLa cells, whereas the
total amount of HuR is unchanged (16). In the present experiments, we found that HuR protein levels were not altered throughout the entire 60-min ischemic period (Fig. 7). This
suggests that in response to acute ischemia and hypoxia,
skeletal muscle HuR expression is not the main mechanism for signaling
VEGF upregulation. Alternatively, the binding activity of HuR to VEGF
mRNA is increased in skeletal muscle after an acute ischemic
bout (Fig. 5). These results suggest that, besides the translocation
mechanism identified in reported in vitro experiments with HeLa cells,
the binding activity of HuR to the mRNA 3'-UTR is also changed in
skeletal muscle during hypoxia, possibly from ligand regulation of HuR. The observation that it requires only a few minutes for an increase in
constitutive HuR binding activity would further suggest that a
hypoxia-induced chemical modification, such as phosphorylation, is
likely to be involved in this mechanism of gene regulation. The
increased binding of HuR to the VEGF mRNA 3'-UTR will ultimately decrease its degradation and increase the level of VEGF mRNA in the
cytoplasm available to the translational machinery.
Possible role of HuR in VEGF regulation at different levels of hypoxia. Many physiological and pathological conditions cause skeletal muscle hypoxia, including exercise, ischemia, and anemia. Even though it is hard to measure skeletal muscle tissue PO2 directly, different methods have been used to reflect this parameter, for example, measurement of venous PO2 (20), multiple channel surface electrode estimation of PO2 (19, 32), and muscle microvascular measurements of PO2 (3). Sjoberg et al. (32) reported that, after blood flow is totally blocked in the rat hindlimb, the muscle surface oxygen levels decrease to ~50% of normal levels in 10 min and approach zero by 20 min. In our experiments, we found that detectable HuR-VEGF 3'-UTR binding increased as early as 5 min after total blood flow was blocked (Fig. 5). This result demonstrates that HuR-VEGF regulation is very sensitive to hypoxia and can be activated at PO2 levels above the 50% of normal values expected if blood flow was restricted for an additional 5 min as in the Sjoberg experiment (32). In addition to ischemic conditions, anemia, exercise, and high altitude have also been reported to decrease PO2 in skeletal muscle. For instance, if the rabbit hemoglobin concentration is decreased to 8.6 g/dl compared with the normal value of 10.8 g/dl, extracellular tissue PO2 in skeletal muscle falls to 47% of control values (19). Furthermore, rat gastrocnemius muscle microvascular PO2 levels decrease to 30% of normal values after 90 s of electrically stimulated muscle contraction (3). Finally, popliteal venous PO2 decreases by 57% when dogs are ventilated with 10% O2-90% N2 (20). In all these conditions, the potential for HuR-VEGF regulation to be a key regulatory step stimulating VEGF expression is high. More carefully controlled experiments under these various physiological conditions are needed to understand the role of HuR proteins in regulating VEGF expression and subsequent new capillary formation in skeletal muscle.
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ACKNOWLEDGEMENTS |
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This research was supported by National Heart, Lung, and Blood Institute Grant HL-17731.
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FOOTNOTES |
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Address for reprint requests and other correspondence: K. Tang, Div. of Physiology, Dept. of Medicine, Univ. of California at San Diego, 9500 Gilman Drive, MC 0623A, La Jolla, CA 92093-0623 (E-mail: ktang{at}ucsd.edu).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
May 23, 2002;10.1152/ajpheart.00813.2001
Received 17 September 2001; accepted in final form 20 May 2002.
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METHODS
RESULTS
DISCUSSION
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, Free-labeled probe that was not
incubated with muscle extracts. In some lanes, 100-fold (100×) or
10-fold (10×) excess nonradioactive probe 59-71 was included in
the reaction mixture and compared with a standard reaction containing
labeled probe 59-71 alone. Nonradioactive probes competitively
inhibited both constitutive and inducible binding activity. A
supershift band is present in 20-min ischemia-exposed samples
incubated with an HuR specific antibody (3A2).
