Validation of optimized RapR-Shp2 phosphatase activity and signaling in living cells. (A) Immunoblot analysis of ERK activation induced by RapR-Shp2 in HEK293T cells. Cells transiently transfected with CA-Shp2-flag, DN-Shp2-flag, and RapR-Shp2-flag constructs as well as nontransfected cells were treated with rapamycin or ethanol (control) as indicated. Lysates were analyzed by immunoblotting using indicated antibodies. Representative blots of N = 3 independent biological replicates. (B) Dephosphorylation of endogenous substrates by RapR-Shp2. A431 cells were transduced with adenoviral RapR-Shp2-mVenus-flag and mCherry-FRB constructs, treated with rapamycin or ethanol (control) for 2 or 4 h, and assessed for phosphorylation of EGFR (pY⁹⁹²), FAK (pY^576/577), PLCγ (pY⁷⁸³), ROCK II (pY⁷²²), and Gab1 (pY⁶²⁷ or pY⁶⁵⁹). Representative blots of N = 5 independent biological replicates. (C) Validation of RapR-Shp2 protein-protein interactions by coimmunoprecipitation. Lentiviral CA-Shp2-mVenus or adenoviral RapR-Shp2-mVenus were expressed in A431 cells, immunoprecipitated, and the presence of Shp2 interacting proteins, EGFR and Gab1, were assessed by immunoblotting. Representative blots of N = 3 (EGFR) and N = 6 (Gab1) independent experiments.