Fig. 7: Y692 served as a self-inhibitory phosphorylation site of PDGFRβ.
From: PTPN14 aggravates neointimal hyperplasia via boosting PDGFRβ signaling in smooth muscle cells
A, B Human aortic smooth muscle cells (HA-VSMCs) were transfected with indicated adenovirus for 48 h, followed by PDGF-BB (10 ng/mL) stimulation for 0, 10, and 30 min. Representative western blots and quantifications of the expression level of indicated proteins. The data were presented as the means ± SEM, n = 6 (two-way ANOVA with Bonferroni multiple comparison post-hoc test). C, D HA-VSMCs were transfected with indicated adenovirus for 48 h, followed by stimulation with PDGF-BB (10 ng/mL) for 10 min. Representative western blots and quantification of the expression level of indicated proteins. The data were presented as the means ± SEM, n = 6 (two-way ANOVA with Bonferroni multiple comparison post-hoc test). E, F PTPN14SMC−/− and the littermate PTPN14flox mice were subjected to wire injury of the left carotid arteries (LCAs) for 21 d. Representative immunofluorescence staining of phosphor-PDGFRβY692 and α-SMA in cross-sections of the LCAs. Quantification of the levels of phosphor-PDGFRβY692. PDGFRβY692, red; α-SMA, green; DAPI, blue. Scale bar, 50 μm. The data were presented as the means ± SEM, n = 6 (two-tailed unpaired Student’s t test). G The binding affinity of PDGF-BB to recombinant protein PDGFRβ and PDGFRβY692D were measured via surface plasmon resonance. H, I Representative immunofluorescence staining of in situ proximity ligation assay for visualization on the interaction between PDGF-BB and PDGFRβ in right carotid arteries (RCAs) and LCAs of PTPN14SMC−/− and PTPN14flox mice 21 d post-injury, respectively. Positive signal of PDGF-BB and PDGFRβ, red; SM22, green; DAPI, blue. Scale bar, 20 μm. Quantification of the positive signal (red) in cross-sections of the RCAs and LCAs. The data were presented as the means ± SEM, n = 6 (two-way ANOVA with Bonferroni multiple comparison post-hoc test). J HA-VSMCs were transfected with the indicated adenoviruses for 48 h, followed by stimulation with PDGF-BB (50 ng/mL) for 30 min. Cells were then incubated with disuccinimidyl suberate (100 μM) at room temperature for 30 min. Representative western blots images of PDGFRβ dimerization were presented, n = 3.
