Figure S5.

FAK is downregulated in vivo compared to in vitro, and there is no change in C3G or ERK recruitment to Paxillin in cells expressing Y118F-Paxillin in vivo. (A) Unmodified Western blot for Fig. 5 B—YUMM1.7 cells plated in culture and YUMM1.7 melanoma tumors (in vivo) blotted with FAK and pY397-FAK antibodies. GFP was used as the loading control, and as a control for the number of YUMM1.7 cells in mouse tumors. (B) YUMM1.7 cells overexpressing GFP-FAK and Western blotted for FAK levels to confirm overexpression in vitro and in vivo. Actin is used as a loading control. (C) Co-immunoprecipitation analysis of Vinculin and Paxillin, immunoprecipitating Paxillin from YUMM1.7 tumors expressing wildtype, Y118E, and Y118F Paxillin and assaying for Vinculin interactions. (D–I) Co-immunoprecipitation analyses of C3G or activated ERK (p-ERK) with Paxillin in YUMM1.7 cell lines that exogenously express mammalian wildtype, Y118E, and Y118F Paxillin in vitro (D–F) and in in vivo tumors (G–I). (E and H) Quantification of C3G/Paxillin ratio from D and G, bands from cells expressing wildtype Paxillin are normalized to 1 both in vitro and in vivo. n = 3 technical replicates. Non-parametric one-way ANOVA, error bars are mean ± SD. (F and I) Quantification of p-ERK/Paxillin levels from D and G, bands from cells expressing wildtype Paxillin are normalized to 1 both in vitro and in vivo. n = 3 technical replicates. Non-parametric one-way ANOVA, error bars are mean ± SD. (J) Western blot analysis of pY31-Paxillin in mouse melanoma YUMM1.7 cells expressing mammalian WT-Paxillin-T2A-GFP plated on the in vitro cell culture dishes (n = 3 dishes) and YUMM1.7 melanoma in vivo tumors (n = 5 tumors). GFP was used as the loading control and a control for number of YUMM1.7 cells in mouse tumors. (K) Quantification of pY31-Paxillin/total Paxillin protein ratio from J. Error bars are mean ± SD. Non-parametric unpaired t test. Source data are available for this figure: SourceData FS5.