![Validation of RapR-Shp2 cellular function. (A) Analysis of Western blot intensity for ERK activation following RapR-Shp2 activation (Fig. 3 A). All phospho-ERK (pT202/pY204) signals are normalized to total ERK blot intensity. One-way ANOVA analysis with post hoc Holm-Bonferroni test for pairwise comparison at a significance level of 0.05. ANOVA F = 7.63, P = 0.001. *P < 0.05. (B) Analysis of the signal intensity for EGFR (pY992) (F[2,1] = 21.45, P < 0.001), FAK (pY576/577) (F[2,1] = 25.35, P < 0.001), PLCγ (pY783) (F[2,1] = 4.56, P = 0.047), ROCK II (pY722) (F[2,1] = 26.23, P < 0.001), Gab1 (pY627) (F[2,1] = 30.87, P < 0.001) or (pY659) (F[2,1] = 42.49, P < 0.001), normalized to corresponding total protein signal, following Rapamycin treatment for 2 or 4 h (Fig. 3 B). Each phosphorylation site was analyzed for significance at the 0.05 level using one-way repeated measures ANOVA with post hoc Dunnett’s method to pairwise compare the fold-change in phosphorylation for each time point relative to the noninfected control group. *P < 0.05, **P < 0.01, ***P < 0.001. (C) Analysis of coimmunoprecipitation of Gab1 and EGFR with FRB-mCherry (non), CA-Shp2-venus, and RapR-Shp2-venus shown in Fig. 4 C (normalized to Shp2). (D–E) Comparison of RapR-Shp2 and endogenous Shp2 expression levels in HEK293T, A431, and HeLa cells. Representative blot of N = 3 independent experiments with quantification (E). All error bars show 90% CI.](https://cdn.rupress.org/rup/content_public/journal/jcb/221/8/10.1083_jcb.202111066/4/jcb_202111066_figs1.png?Expires=1772587145&Signature=Y6ILYhsldmTKUcfJttADWV0-lVWaLhVwVoXR4wlO7CQVYv9iZJ1-hx64NuPMm9oVe6qay3sDE4nyXwKf2fErBFqihytRDLZd30xMZMDrS8UWvqSo99AYJJ4G3JISy5mCcvQc07IgMR-j2u1Da5cEFUgDSAEJQQ0racNg0uBPR4JlAD~pi5VB8fU3cN8JkrXRPvRv4Dh7whA0U-HKJfk5LpFv1Dcm0ejmmGs~AnFLRywNoXU-qETJRbWzeH6huVKZAmncRiCC8Zz-9CrbBLjRPxUSX9P1K4KVOzLqIVNOc0wSSnvKEQeXcXzklhL6RBup5PWshQdrQevysHBGj9hryQ__&Key-Pair-Id=APKAIE5G5CRDK6RD3PGA)
Validation of RapR-Shp2 cellular function. (A) Analysis of Western blot intensity for ERK activation following RapR-Shp2 activation (Fig. 3 A). All phospho-ERK (pT202/pY204) signals are normalized to total ERK blot intensity. One-way ANOVA analysis with post hoc Holm-Bonferroni test for pairwise comparison at a significance level of 0.05. ANOVA F = 7.63, P = 0.001. *P < 0.05. (B) Analysis of the signal intensity for EGFR (pY992) (F[2,1] = 21.45, P < 0.001), FAK (pY576/577) (F[2,1] = 25.35, P < 0.001), PLCγ (pY783) (F[2,1] = 4.56, P = 0.047), ROCK II (pY722) (F[2,1] = 26.23, P < 0.001), Gab1 (pY627) (F[2,1] = 30.87, P < 0.001) or (pY659) (F[2,1] = 42.49, P < 0.001), normalized to corresponding total protein signal, following Rapamycin treatment for 2 or 4 h (Fig. 3 B). Each phosphorylation site was analyzed for significance at the 0.05 level using one-way repeated measures ANOVA with post hoc Dunnett’s method to pairwise compare the fold-change in phosphorylation for each time point relative to the noninfected control group. *P < 0.05, **P < 0.01, ***P < 0.001. (C) Analysis of coimmunoprecipitation of Gab1 and EGFR with FRB-mCherry (non), CA-Shp2-venus, and RapR-Shp2-venus shown in Fig. 4 C (normalized to Shp2). (D–E) Comparison of RapR-Shp2 and endogenous Shp2 expression levels in HEK293T, A431, and HeLa cells. Representative blot of N = 3 independent experiments with quantification (E). All error bars show 90% CI.
