FIGURE 4:

SmgGDS as a novel partner of BPGAP1 and suppressor of K-Ras signaling. (A) HEK293T cells were transfected with Flag-BPGAP1, vector, or BCH, made quiescent, and stimulated with 100 ng/ml of EGF. Lysates were obtained at indicated time points, subjected to immunoprecipitation (IP), and analyzed by Western blotting. Endogenous SmgGDS, bound to overexpressed BPGAP1 and BCH, was detected with anti-SmgGDS, and anti-Flag revealed equal loading of IP beads. (B) Efficiency of knockdown shRNA constructs rS1 and rS2 targeting SmgGDS were tested by transfecting HEK293T cells with shRNA constructs and HA-SmgGDS in a ratio of 1:10, and lysates were used to detect levels of SmgGDS (top panel). Tubulin was detected as a loading control (bottom panel). (C) Knockdown of SmgGDS increases K-Ras activation. HEK293T cells were transfected with pSilencer negative control or SmgGDS targeting rS1 for 48 h, made quiescent, and subjected to EGF stimulation at 100 ng/ml. Lysates obtained at indicated time points were subjected to the K-Ras activation assay.