Fig. 6: PTPN14 interacted with PDGFRβ and mediated its dephosphorylation at Y692 site.

A Representative western blots of PDGFRs immunoprecipitated using PTPN14 antibody in human aortic smooth muscle cells (HA-VSMCs). n = 3. B HA-VSMCs were treated with PDGF-BB (20 ng/mL) or the PBS control for 10 min. Confirmation of the interaction between PDGFRβ and PTPN14 via western blot analysis. n = 3. C, D The co-immunoprecipitation assay to confirm the interaction with co-transfected Flag-tagged PTPN14 (Flag-PTPN14) and HA-tagged PDGFRβ (HA-PDGFRβ) in HEK293T. n = 3. E Schematic diagram of plasmid truncations construction of PDGFRβ. The positions of the amino acid residues of each structural domain were labeled. F The immunoprecipitation assay was performed to detect the interaction among PTPN14 and various domain fragments of PDGFRβ with Flag-PTPN14 and HA-PDGFRβ domains in HEK293T. n = 3. G HA-VSMCs were transfected with control (siNC) or PTPN14 (siPTPN14) small interfering RNA, then treated with adenovirus to overexpress PDGFRβ for 48 h. After stimulation with PDGF-BB (10 ng/mL) for 10 min, cells were harvested for phosphorylation site detection. Mass spectrometry analysis profile of PDGFRβ dephosphorylated by PTPN14 at Y692. n = 3. H–I HA-VSMCs were transfected with siNC or siPTPN14, then treated with PDGF-BB (10 ng/mL) for indicated time. Representative western blots and quantification of PDGFRβ phosphorylation level at Y692. The data were presented as the means ± SEM, n = 6 (two-way ANOVA with Bonferroni multiple comparison post-hoc test).