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. 2015 Mar 10;290(18):11403–11416. doi: 10.1074/jbc.M115.645788

FIGURE 2.

FIGURE 2.

Expression of VASP S157A in ASM tissues inhibits ACh-stimulated contractile force and actin polymerization, but it does not affect MLC phosphorylation. A, active stress in ASM tissues in response to stimulation with 10−5 m ACh before and after expression of His-EGFP VASP WT, His-EGFP VASP S157A, or sham transfection. B, expression of His-EGFP VASP S157A significantly depressed the force relative to sham-treated tissues, but expression of His-EGFP VASP WT had no significant effect. ACh-stimulated force normalized to maximal force in sham-treated tissues. *, significantly different from sham-treated tissues (n = 9). C, immunoblot against the His epitope of VASP immunoprecipitates from ACh-stimulated or unstimulated (US) tissues expressing His-EGFP VASP WT, His-EGFP VASP S157A, or sham treated. Expression of recombinant His-EGFP VASP and His-EGFP VASP S157A was observed in transfected tissues but not sham-treated tissues. D, immunoblot against VASP and VASP phospho-Ser157 from tissues transfected with His-EGFP VASP WT, His-EGFP VASP S157A, or sham treated. E, expression of VASP S157A significantly inhibited endogenous VASP Ser157 phosphorylation in response to 10−5 m ACh stimulation (n = 11 for sham and VASP S157A, n = 6 for VASP WT). *, significantly different from sham-treated tissues. F, actin polymerization was determined in tissues expressing VASP S157A, VASP WT, or sham treated. Actin polymerization increases in response to ACh in sham-treated and VASP WT-treated muscle tissues but not in tissues expressing VASP S157A. *, significantly different (n = 11 for sham and VASP S157A, n = 5 for VASP WT). G, actin polymerization was determined in tissues stimulated with ACh, FSK, or US. Actin polymerization was significantly increased in response to ACh stimulation but not with FSK stimulation (n = 4). H and I, MLC phosphorylation measured by urea gel electrophoresis in US or ACh-stimulated tissues expressing VASP S157A, VASP WT, or sham treated. There were no significant differences in MLC phosphorylation among ACh-stimulated or US tissues subjected to different treatments (n = 5).