Molecular Characterization of Striated Muscle-Specific Gab1 Isoform as a Critical Signal Transducer for Neuregulin-1/ErbB Signaling in Cardiomyocytes

Grb2-associated binder (Gab) docking proteins regulate signals downstream of a variety of growth factors and receptor tyrosine kinases. Neuregulin-1 (NRG-1), a member of epidermal growth factor family, plays a critical role for cardiomyocyte proliferation and prevention of heart failure via ErbB receptors. We previously reported that Gab1 and Gab2 in the myocardium are essential for maintenance of myocardial function in the postnatal heart via transmission of NRG-1/ErbB-signaling through analysis of Gab1/Gab2 cardiomyocyte-specific double knockout mice. In that study, we also found that there is an unknown high-molecular weight (high-MW) Gab1 isoform (120 kDa) expressed exclusively in the heart, in addition to the ubiquitously expressed low-MW (100 kDa) Gab1. However, the high-MW Gab1 has been molecularly ill-defined to date. Here, we identified the high-MW Gab1 as a striated muscle-specific isoform. The high-MW Gab1 has an extra exon encoding 27 amino acid residues between the already-known 3rd and 4th exons of the ubiquitously expressed low-MW Gab1. Expression analysis by RT-PCR and immunostaining with the antibody specific for the high-MW Gab1 demonstrate that the high-MW Gab1 isoform is exclusively expressed in striated muscle including heart and skeletal muscle. The ratio of high-MW Gab1/ total Gab1 mRNAs increased along with heart development. The high-MW Gab1 isoform in heart underwent tyrosine-phosphorylation exclusively after intravenous administration of NRG-1, among several growth factors. Adenovirus-mediated overexpression of the high-MW Gab1 induces more sustained activation of AKT after stimulation with NRG-1 in cardiomyocytes compared with that of β-galactosidase. On the contrary, siRNA-mediated knockdown of the high-MW Gab1 significantly attenuated AKT activation after stimulation with NRG-1 in cardiomyocytes. Taken together, these findings suggest that the striated muscle-specific high-MW isoform of Gab1 has a crucial role for NRG-1/ErbB signaling in cardiomyocytes.

muscle-specific high-MW isoform of Gab1 has a crucial role for NRG-1/ErbB signaling in cardiomyocytes.
MW Gab1 has been elusive to date. Here we identified a novel high-MW Gab1 which is exclusively expressed in striated muscle, and characterized it as a crucial signaling component for the activation of PI3-kinase/AKT signaling axis downstream of NRG-1/ErbB signaling in cardiomyocytes.

Animals
All experiments were carried out under the guidelines of the Osaka University Committee for Animal and Recombinant DNA Experiments and the local Animal Ethics Committee of the National Cerebral and Cardiovascular Center Research Institute (Osaka). All experiments were also approved by the Institutional Review Board of Osaka University and the National Cerebral and Cardiovascular Center Research Institute. Male C57BL/6J 8-week(wk)-old mice and timed-pregnant C57BL/6J mice, purchased from Japan SLC, Inc., were used in the experiments.

Immunoprecipitation and western blot analysis of mouse tissues
About 80 mg tissues were excised from male C57BL/6J 8-wk-old mouse and snap-frozen in liquid nitrogen. Frozen heart, brain, spleen, lung, liver, and skeletal muscle were homogenized in 50 mmol/L Hepes, 100 mmol/L sodium fluoride, 2 mmol/L sodium orthovanadate, 4 mmol/L EDTA, 1% Tween-20, 0.1% SDS, and Complete protease inhibitor cocktail (Roche Applied Science) using Polytron homogenizer (PT 10-35GT; Kinematica) as described previously [22,34]. The lysates were precleared by centrifugation at 15,000 × g for 15 minutes (min). For immunoprecipitation, the cleared lysates were rotationally incubated with either 1 μL of anti-Gab1 or 2 μL of anti-high MW Gab1 serum and with 20 μL of protein A-sepharose (GE Healthcare) for 8 hours (hr) at 4˚C. The antigen-antibody complexes were collected by centrifugation, washed three times with lysis buffer without protease inhibitor mixture, and boiled in standard electrophoresis sample buffer. All the immunoprecipitated proteins were then resolved by SDS-PAGE and subjected to immunoblotting using standard procedure. Blots were developed using ECL system (GE Healthcare).

Sequencing of mouse Gab1 Extra Exon
Total RNA was isolated from adult mouse heart, lung, and brain using TRIzol (Invitrogen). cDNA was synthesized using SuperScript II Reverse Transcriptase (Invitrogen) and subjected to PCR (94˚C for 2 min; 25 cycles through 94˚C for 30 seconds (sec), 60˚C for 30 sec, 72˚C for 90 sec, and then extension at 72˚C for 10 min) using the forward primer 5'-CCTGTGAAGCCG CTGACTGGCTC-3' located in exon 3 and the reverse primer 5'-TTTCCGCAACTTGTTCAA ATCCACGG-3' located in exon 4. PCR products were electrophoresed in a 1.5% agarose gel. Double bands were detected only in the cDNA purified from heart, and the higher band was purified using QIAEX II Agarose Gel Extraction Kit (QIAGEN). The purified PCR products were cloned using Zero Blunt TOPO PCR Cloning Kit for Sequencing (Invitrogen) and transformed into JM109 competent cells. Plasmid DNA was extracted and sequenced. The sequence was analyzed using BLAST.
Cloning of the full-length mouse Gab1 cDNA Total RNA was isolated from adult mouse heart using TRIzol. cDNA was synthesized using SuperScript II Reverse Transcriptase (Invitrogen) and subjected to PCR (94˚C for 30 sec; 35 cycles through 94˚C for 15 sec, 72˚C for 3 min, and then extension at 72˚C for 2 min) using the forward primer 5'-GCGGATCCATGAGCGGCGGCGAAGTG-3' and the reverse primer 5'-CGGCGGCCGCTGGCTCGAGGCTCCACTGAGA-3'. Purified and restriction-digested products were inserted into pcDNA3 at BamHI and NotI restriction sites. pcDNA3 constructs of mouse Gab1 were transformed into JM109 competent cells. Plasmid DNA was extracted and subjected to PCR (94˚C for 3 min; 30 cycles through 94˚C for 30 sec, 60˚C for 30 sec, 72˚C for 40 sec, and then extension at 72˚C for 4 min) using the forward primer 5'-CAAAGCAAGA AGCCTGAACC-3' located in exon 3 and the reverse primer 5'-CTCTCCCGAGACAGATGTC AG-3' located in exon 4. The predicted amplified fragments of mouse high-and low-MW Gab1 were 280 and 200 base pairs, respectively. Plasmid DNA which was thought to contain mouse high-or low-MW Gab1 was sequenced.
Transfection and western blot analysis pcDNA3 constructs of mouse high-or low-MW Gab1 was transfected into HEK293 cells with Lipofectamine 2000 according to the manufacturer's instructions (Invitrogen). After 4 hr, the cells were returned to growth medium and incubated for 48 hr. Cells were washed twice with ice-cold PBS and scraped off in lysis buffer containing 20 mmol/L Tris (pH 7.4), 150 mmol/L NaCl, 3 mmol/L EDTA, 1% Nonidet P-40, 2 mmol/L orthovanadate and Complete protease inhibitor cocktail as described previously [22,23,32]. Cell lysates were precleared by centrifugation at 15,000 × g for 15 min. The cleared lysates were subjected to immunoblot analysis following standard procedures as described above.

Expression analysis using cDNA panel
Mouse MTC panel I (Clontech) was utilized for the analysis of high-MW Gab1 expression pattern. The MTC cDNA was subjected to PCR (94˚C for 3 min; 32 cycles through 94˚C for 30 sec, 60˚C for 30 sec, 72˚C for 1 min, and then extension at 72˚C for 7 min) using the forward primer 5'-CCAAGGGTCATCTTTTGTTTC-3' located in extra exon or 5'-CAAAGCAAGAA GCCTGAACC-3' located in exon 3 and the reverse primer 5'-CTCTCCCGAGACAGATGTCA G-3' located in exon 4. PCR products were electrophoresed in a 1.5% agarose gel.

Immunohistochemical analysis of mouse tissues
For immunohistochemial analysis, anti-high-MW Gab1 antiserum was absorbed with mouse liver and kidney powder as described previously [36]. Mouse liver and kidney powder was prepared from cold acetone homogenate [36]. Mouse liver and kidney powder was washed in PBS and used to absorb antiserum diluted 1:4 in 0.2% BlockAce (DS Pharma Biomedical). The mixture was shaken overnight at 4˚C. The absorbed antiserum was separated from acetone powder by centrifugation at 10,000 × g for 10 min. Embryos were embedded in OCT (Sakura) for frozen sections and cut by cryosectioning (10 μm) [35]. Sections were fixed in pre-cooled acetone for 10 min. Sections were incubated in blocking solution containing 1% bovine serum albumin in PBS and 0.1% Triton-X (PBST) for 1 h at room temperature and then incubated with primary antibodies in PBST containing 1% bovine serum albumin overnight at 4˚C. The following primary antibodies were used for immunostaining: absorbed anti-high MW Gab1 (1:10 dilution); anti-α-Actinin (1:300 dilution). Sections were then washed in PBST and incubated with fluorescent-conjugated (1:200 dilution; Alexa Fluor 488 and 546; Invitrogen) secondary antibodies for 1 hr at room temperature. Images were acquired with a confocal microscope (LSM710; Zeiss).

Biochemical analysis in vivo
Biochemical analysis of mice hearts after stimulation with various growth factors and cytokines was performed as described previously [22]. Briefly, mice anesthetized by isoflurane were injected i.v. via the inferior vena cava with following agonists: LIF (1×10 4 U), NRG-1β (5 μg), HB-EGF (5 μg), and EGF (5 μg). The hearts were isolated at 5 min after injection and washed with ice-cold PBS. After removing both atria, the ventricles were snap-frozen in liquid nitrogen. Immunoprecipitation and western blot analysis were performed as described above. Band intensity was quantified by densitometry.

Adenovirus vector construction
The cDNA sequence encoding mouse high-and low-MW Gab1 was subcloned into the shuttle plasmid pACCMVpLpA. Recombinant adenovirus was then obtained according to the homologous recombination system described previously [32,37]. The adenovirus vector expressing β-gal was used as a control.
After serum-starvation in medium 199 for 24 h, NRCMs were stimulated with 20 ng/mL NRG-1β for the indicated periods. Cells were lysed, precleared, and subjected to immunoprecipitation and immunoblot analysis following standard procedures as described earlier.

Statistical analysis
Mean values, standard error of measurement, and statistical analysis for all data shown were calculated and plotted using GraphPad Prism 5 (GraphPad Software). All data were expressed as mean ± SEM. Differences among multiple groups were compared by one-way ANOVA followed by a post hoc comparison tested with Tukey's method. A value of P < 0.05 was considered as statistically significant.

High-MW Gab1 is specifically expressed in the heart
To address the expression pattern of Gab1 in the various organs in mice, we performed immunoprecipitation and immunoblot analysis using the protein extracts from heart, brain, spleen, lung, liver, and skeletal muscle from C57BL/6J 8-wk old male mouse. Although low-MW Gab1 was detected in all the organs examined, high-MW Gab1 was exclusively expressed in the heart (Fig 1A). This finding is consistent with the data which we reported previously [22].
High-MW Gab1 cDNA contains one skipped exon encoding highly conserved 27 amino acid residues We hypothesized that a skipped extra exon exists in the high-MW Gab1 cDNA. We systematically searched for an extra exon between the already-known exons of the low-MW Gab1 cDNA by comparing the RT-PCR products derived from the total RNAs of various organs. When using the forward primer in exon 3 and the reverse primer in exon 4, we found two distinct RT-PCR products in the RNAs from heart, but not in those from brain and lung (Fig 2A). On the other hand, we could not detect other extra exons in the heart RNA when using other combination of primers targeting other exons (S1 Fig and S1 Table). We found that a skipped extra exon between the already-known exon 3 and the exon 4 consists of 81 base pairs and encodes 27 amino acid residues (Fig 2B and 2C). Intriguingly, the 27 amino acid sequence encoded by this extra exon is highly conserved in mammals, suggesting that it might have a role for cardiomyocytes ( Fig 2D). However, the 27 amino acid sequence does not show homology to the already-known functional domains.
We next isolated the mouse full-length cDNA (high-MW Gab1) containing the extra exon. HEK293 cells were transfected with the pcDNA3 vector containing the cDNAs encoding the low-MW and the high-MW Gab1, and we confirmed that the expected difference in electrophoretic mobility between the high-MW Gab1 and the low-MW Gab1 by western blot analysis (Fig 2E).

High-MW Gab1 might be a novel striated muscle-specific Gab1 isoform
To determine the tissue expression pattern of the high-MW Gab1, we performed RT-PCR analysis using the cDNA panel derived from several adult mouse tissues and mouse embryos at various developmental stages. We performed two types of RT-PCR, using (1) the primers targeting the extra exon exclusively, and (2) the primers targeting the exon 3 and the exon 4. Both experiments showed that high-MW Gab1 was most strongly expressed in the adult heart and barely detected in adult skeletal muscle (Fig 3A). We next examined the expression level of Gab1 in mice hearts from embryonic to adult stage. Although the expression levels of high-MW Gab1 and Gab1 (including both of high-and low-MW Gab1) mRNA were not significantly different between embryonic and adult hearts, the high-MW Gab1 mRNA to total Gab1 mRNA ratio significantly increased along with heart development (Fig 3B-3D). We also performed immunoprecipitation and immunoblot analysis using the protein extracts from embryonic, postnatal, and adult mice hearts. Consistent with the qRT-PCR data, the high-MW Gab1 protein was expressed in the same level from embryonic to adult stage, although the expression level of the low-MW Gab1 protein decreased along with heart development ( Fig 3E).
Next, we raised rabbit polyclonal antibody specific for the high-MW Gab1. Immunostaining with this antibody in E11.5 embryos showed that the high-MW Gab1 was expressed in heart, but not in liver (Fig 4A). E17.5 embryos also showed that the high-MW Gab1 was expressed in heart and skeletal muscle, but not in lung (Fig 4B). We also performed immunoprecipitation and immunoblot analysis using the protein extracts from C57BL/6J 8-wk old male mice. The high-MW Gab1 was immunoprecipitated with this antiserum exclusively in the lysate extracted from heart, but not from other organs including skeletal muscles of adult mice (Fig 1B). These findings suggest that the high-MW Gab1 might be a novel striated muscle-specific Gab1 isoform.

Fig 2. High-MW Gab1 contains extra-exon coding 27 amino acid residues.
A) Mouse heart, lung, and brain RNA were subjected to RT-PCR using the forward primer to exon 3 (rightwards arrow) and the reverse primer to exon 4 (leftwards arrow). The long fragment (arrowhead) was detected in heart, but not in lung and brain. High-MW Gab1 undergoes tyrosine-phosphorylation in the murine heart after intravenous administration of NRG-1 We previously reported that the high-MW Gab1 underwent tyrosine-phosphorylation exclusively upon stimulation with NRG-1 among several growth factors and cytokines [22]. To confirm the tyrosine-phosphorylation of the high-MW Gab1 in the adult murine hearts, we intravenously administrated several humoral factors such as leukemia inhibitory factor (LIF), NRG-1, HB-EGF, and EGF into wild-type mice. We subjected the heart lysates to immunoprecipitation with anti-high-MW Gab1 and western blot analysis. Consistent with previous data, the high-MW Gab1 underwent tyrosine-phosphorylation exclusively after administration of NRG-1, but not with LIF, HB-EGF, and EGF (Fig 5A) [22]. We also checked the activation of ErbB4. NRG-1 exclusively induced tyrosine-phosphorylation of ErbB4 (Fig 5B). Whereas . Values are shown as mean±SEM for 3 separate experiments. One-way ANOVA followed by Tukey's test was used to analyses differences. D) Quantitative expression analysis of high-MW Gab1 to Gab1 mRNA ratio in hearts at E11.5, E18.5, P7, and P28 (n = 3). Values are shown as mean±SEM for 3 separate experiments. One-way ANOVA followed by Tukey's test was used to analyses differences. E) Expression patterns of high-MW Gab1 (arrowhead) and low-MW Gab1 (arrow) protein in mouse hearts at E18.5, P7, P28, and P56. Following immunoprecipitation with anti-Gab1 serum, expression of Gab1 was examined by immunoblotting analysis with anti-Gab1 antibody. SHP2 was examined as a loading control. Representative blots of 2 experiments are shown. activation of ERK1/2 was almost comparable in the mice treated with NRG, HB-EGF (Fig 5C  and 5D), and EGF, activation of AKT was most strongly induced by NRG-1 (Fig 5C and 5E).
High-MW Gab1 plays a crucial role in AKT activation downstream of NRG-1/ErbB signaling Next, we examined the role of the high-MW Gab1 in NRG-1/ErbB signaling. Since PI3-kinase pathway has been reported to have critical roles for the proliferation of the cardiomyocytes and protection by NRG-1/ErbB signaling [15,39], we focused on the activation of AKT after The Roles of Striated Muscle-Specific Gab1 Isoform in Neuregulin-1/ErbB Signaling stimulation with NRG-1 in cardiomyocytes. First, we examined the effect of adenovirus-mediated forced expression of high-MW Gab1 on the NRG-1-dependent downstream signaling pathways. Overexpression of the high-MW Gab1 induced much stronger association of Gab1 with p85 than that with SHP2 upon stimulation with NRG-1 (Fig 6A). NRG-1 induced activation of AKT and ERK1/2 in the control NRCMs expressing β-galactosidase (β-gal) (Fig 6B). Whereas NRG-1-induced activation of ERK1/2 was not significantly changed in NRCMs expressing the high-MW Gab1 compared with control cells expressing β-gal (Fig 6B and 6C), activation of AKT was significantly enhanced in NRCMs expressing the high-MW Gab1 compared with control cells expressing β-gal (Fig 6B and 6D). In addition, NRG-1-induced activation of AKT was also enhanced in NRCMs expressing the low-MW Gab1 compared with control cells expressing β-gal, whereas activation of ERK1/2 was not significantly changed in NRCMs expressing the low-MW Gab1 compared with control cells expressing β-gal (S2 Fig).   Fig 5. High-MW Gab1 is exclusively activated after stimulation with NRG-1 in heart. A) Tyrosine phosphorylation of the high-MW Gab1 was analyzed by immunoprecipitation of the heart lysates. Mouse heart lysates were prepared at 5 min after injection with the cytokines and growth factors listed at top. Heart lysates were subjected to immunoprecipitation with anti-high-MW Gab1 serum, followed by western blot analysis using the antibodies indicated at the left. Arrowheads denote the high-MW Gab1. B) Activation level of ErbB4 was assessed by phospho-specific antibody. C) Activation levels of ERK1/2 and AKT were assessed by phospho-specific antibodies. Heart lysates were subjected to western blot analysis with the indicated antibodies. Representative blots of 3 experiments are shown. D) Phosphorylation of ERK1/2 was quantified against total ERK1/2 (n = 3). E) Phosphorylation of AKT on Ser-473 was quantified against total AKT (n = 3). Values are shown as means±SEM for 3 separate experiments. One-way ANOVA followed by Tukey's test was used to analyze differences. *P<0.05, **P<0.01, and ***P<0.001 for the indicated groups. The Roles of Striated Muscle-Specific Gab1 Isoform in Neuregulin-1/ErbB Signaling These findings suggest that the high-MW shares a role for enhancing the NRG-1-induced activation of AKT in NRCMs with the low-MW Gab1 isoform.
Next, we performed siRNA-mediated knockdown of high-MW Gab1 in NRCMs. We observed successful depletion of the high-MW Gab1 protein in NRCMs at 72 hr after transfection with the high-MW Gab1-specific siRNA (Fig 7A). SiRNA-mediated knockdown of the high-MW Gab1 significantly attenuated sustained activation of AKT in response to NRG-1 in NRCMs (Fig 7A and C), but had no obvious effects on activation of ERK1/2 in response to NRG-1 (Fig 7A and 7B). Taken together, these results suggest that the high-MW Gab1 has a crucial role for AKT activation after stimulation with NRG-1 in NRCMs.

Discussion
In the present study, we identified a novel isoform of Gab1 (high-MW Gab1). This high-MW Gab1 has a skipped exon coding 27 amino acid residues between the already-known 3 rd and 4th exons of the ubiquitously expressed low-MW Gab1. Whereas the high-MW Gab1 is  expressed in both heart and skeletal muscle in embryos, it is exclusively expressed in heart during adulthood. The high-MW Gab1 in the heart undergoes tyrosine-phosphorylation only when stimulated with NRG-1. Forced expression of the high-MW Gab1 induces higher and more sustained activation of AKT after stimulation with NRG-1 compared with that of βgalactosidase in cardiomyocytes. Furthermore, knockdown of the high-MW Gab1 significantly attenuated NRG-1-induced activation of AKT in cardiomyocytes. These data indicate that the high-MW Gab1 isoform expressed in striated muscles might have a crucial role for activation of PI3-kinase/AKT pathway downstream of NRG-1/ErbB signaling in cardiomyocytes.
To our knowledge, there has been no report regarding a striated muscle-specific isoform of docking proteins. We identified a novel high-MW isoform (120 kDa) of Gab1 expressed exclusively in the striated muscles such as cardiac and skeletal muscles, in addition to the ubiquitously expressed low-MW (100 kDa) Gab1. In adult mice, the low-MW Gab1 is expressed ubiquitously in any cell types and in any organs [16][17][18]31]. It was reported that the expression of Gab1 is confined to myocardium from embryonic day 10.5 (E10.5) to E13.5, and becomes ubiquitous after E13.5 along with development [20]. The high-MW Gab1 isoform might contribute to the myocardium-specific expression pattern of Gab1 in midterm embryos and might be related to the cardiac defect in conventional Gab1KO mice [20].
Although Gab1 is a common docking protein mediating the signaling from diverse growth factors and cytokines, the high-MW Gab1 specifically underwent tyrosine-phosphorylation upon stimulation with NRG-1 (Fig 5A). We previously reported that intravenous administration of NRG-1 into C57BL6 mice induced tyrosine-phosphorylation of ErbB2 and ErbB4, but not that of ErbB1 and ErbB3 in the hearts [22]. In addition, we also showed that Gab1 associated with ErbB4 in NRG-1-dependent manner [22]. In the present study, we found that NRG-1 exclusively induced tyrosine-phosphorylation of ErbB4 among LIF, NRG-1, HB-EGF, and EGF ( Fig 5B). These data indicate that NRG-1/ErbB4 signaling might specifically utilize Gab1 for activation of downstream pathways.
Forced expression of the high-MW Gab1 enhances and prolongs AKT activation after stimulation with NRG-1 in cardiomyocytes (Fig 6B-6D). On the other hand, overexpression of the low-MW Gab1 also enhances AKT activation after stimulation with NRG-1 in cardiomyocytes almost similarly as that of the high-MW Gab1 (S2 Fig). Whereas knockdown of the high-MW Gab1 significantly attenuated NRG-1-induced activation of AKT in cardiomyocytes (Fig 7), it was technically impossible to perform selective knockdown of the low-MW Gab1 in cardiomyocytes. Therefore, we could not exclude the possibility that the high-MW Gab1 and the low-MW Gab1 might share a similar role for activation of AKT downstream of NRG-1/ErbB signaling in cardiomyocytes.
In the previous study, we reported that cardiomyocyte-specific Gab1 knockout (Gab1CKO) was not sufficient to attenuate the activation of AKT upon NRG-1 administration [22]. The attenuation of AKT activation could only be achieved by Gab1 and Gab2 double knockout (DKO) [22]. For the creation of Gab1CKO, we crossed alpha-MHC-Cre transgenic mice with Gab1flox mice. In Gab1CKO and DKO mice, Gab1 protein expression was deleted in the cardiomyocytes from the embryonic stage, suggesting that some compensatory mechanisms might work. On the other hand, in the present study, we performed acute siRNA-mediated knockdown targeting high-MW Gab1 and found that the high-MW Gab1 knockdown attenuated the activation of AKT upon stimulation with NRG-1 in the neonatal rat cardiomyocytes. Therefore, the above discrepancy might be derived from the difference in experimental condition. Recent studies reported that Gab1CKO mice showed cardiac dysfunction under stressed condition such as myocardial ischemia/reperfusion injury and acute pressure overload with transaortic constriction [27,40]. Our findings implicate the possibility that Gab1CKO mice might show cardiac dysfunction under stressed condition due to impaired response to NRG-1.
In summary, we identified a novel high-MW Gab1 isoform which is exclusively expressed in heart and skeletal muscle, and characterized it as a crucial component for AKT activation downstream of NRG-1/ErbB-signalng in cardiomyocytes.
Supporting Information S1 Fig. No other extra-exon was detected in the heart RNA. When we performed RT-PCR using forward primer to exon 1 and reverse primer to exon 3, forward primer to exon 4 and reverse primer to exon 5, or forward primer to exon 5 and reverse primer to exon 10, one fragment was detected in the heart, brain and lung total RNA. PCR primers used in S1 Figure were summarized in S1 Table. (TIF)

S2 Fig. Forced expression of low-MW Gab1 enhances activation of AKT in response to NRG-1 in NRCMs.
A) Phosphorylation of Gab1, ERK1/2, and AKT were assessed by phosphor-specific antibodies. Representative blots of 5 experiments are shown. B) Phosphorylation of ERK1/2 was quantified against total ERK1/2 (n = 5). C) Phosphorylation of AKT on Ser-473 was quantified against total AKT (n = 5). Values are shown as means±SEM for 5 separate experiments. One-way ANOVA followed by Tukey's test was used to analyze differences. Ã P<0.05, ÃÃ P<0.01, ÃÃÃ P<0.001 for the indicated groups. §P<0.05 vs β-gal expressing cells at the same time after stimulation. (TIF) S1 Table. Primer sequences used in S1 Fig.  (TIF)