Fig. 6

NCK is essential for PDGF-B-induced pericyte migration in vitro. a Schematic of PDGFRβ downstream signaling. NCK adaptor proteins bind the phospho-Y1009 on the PDGFRβ receptor to regulate PDGF-B-induced PAK activation, while other downstream effectors bind to different phosphosites. b qPCR measurement of NCK1 and NCK2 levels in HBMEC (n = 6), human brain SMC (n = 6), and HBVPC (n = 6) compared to HUVEC (n = 5). Error bars represent s.e.m. ^⋆P < 0.05, ^⋆⋆⋆P < 0.001, two-way ANOVA with Sidak’s multiple comparisons test. c qPCR analysis of NCK1, NCK2, and PDGFRβ expression in HBVPC transfected with siRNAs against NCK1 and NCK2 or PDGFRβ compared to control siRNA (siCT) (n = 4 experiments). Error bars represent s.e.m. d Scratch-wound migration assay using HBVPC treated with siRNAs and stimulated with recombinant proteins as indicated for 6 h. Top panels show the wound edges at 0 h. Dashed lines mark wound migration edges at 0 h (straight lines) and 6 h. e Quantification of wound closure (n = 4 experiments). Error bars represent s.e.m. ^⋆⋆P < 0.01, Student’s t test. f Proliferation assay using HBVPC transfected with control (siCT), NCK1 and NCK2 or PDGFRβ siRNAs and stimulated with PDGF-B (n = 3 experiments). Error bars represent s.e.m. ^⋆⋆P < 0.01, Student’s t test. g Western blot using HBVPC stimulated with PDGF-B and probed with the indicated antibodies. h Quantifications of blots shown in g (n = 3 experiments). Error bars represent s.e.m. ^⋆⋆P < 0.01, Student’s t test. Scale bars, 150 μm (d)