Protrusion and Retraction analysis for RapRTAP-Shp2. HeLa cells transiently expressing R32L RapR-Shp2-cerulean-flag and the FRB fused target protein were stained with CellMask Deep Red membrane dye and imaged live, data from Fig. 8. CellMask images were used to determine protrusive and retraction activity binned over 10 min intervals with mean activity at each 2 min time point overlayed as a solid line, error bars show 90% CI. Addition of Rapamycin at time 0 min. Analysis of statistical significance at the 0.05 level was determined using one-way repeated measures ANOVA with Dunnett’s correction relative to the 0 min time point post hoc. (A and B) Analysis of changes in Protrusive (A) and Retraction (B) activity induced by targeted activation of RapR-Shp2 in complex with Gab1 showed significant changes in protrusion (F[23,1] = 9.0, P < 0.001) from time points 10–50 min, 70–120, 140 and 160 min and significant changes in retraction (F[23,1] = 14.04, P < 0.001) from time points 40–130 and 150–180 min. (C and D) Analysis of changes in Protrusive (C) and Retraction (D) activity induced by targeted activation of RapR-Shp2 in complex with Gab2 showed significant changes in protrusion (F[23,1] = 28.29, P < 0.001) from time points 10–180 min and significant changes in retraction (F[23,1] = 40.46, P < 0.001) from time points 20–180 min. (E and F) Analysis of changes in Protrusive (E) and Retraction (F) activity induced by targeted activation of RapR-Shp2 in complex with FAK showed significant changes in protrusion (F[23,1] = 8.95, P < 0.001) from time points 10 and 30–50 min and no significant changes in retraction (F[23,1] = 1.33, P = 0.139). (G–I) Representative images HeLa cells stained with CellMask Deep Red and transfected with R32L RapR-Shp2-cerulean (Shp2) and indicated FRB fusion proteins before and after rapamycin treatment. Focal adhesions with localized Shp2 are indicated with blue arrows.