Figure 4
From: SUMOylation of Grb2 enhances the ERK activity by increasing its binding with Sos1
SUMOylation of Grb2 promotes migration and tumorigenesis by upregulation of the ERK activities. (A-B) Endogenous Grb2 in the murine fibroblast cell line NIH/3T3 (A) and the murine colon cancer cell line CMT-93 (B) was knockdown by a short hairpin RNA targeting Grb2 3′UTR (shGrb2) by using the lentiviral vector pLKO.1 system. Grb2WT and Grb2K56R were reintroduced respectively into these stable Grb2-silencing NIH/3T3 and CMT-93 cells by the lentiviral system. (C) The ERK activities in stable NIH/3T3 cell lines treated with 5 μg/ml of Doxycycline for 24 or 48 hours were determined by Western blotting. (D) Serum-starved stable CMT-93 cell lines were stimulated with 100 ng/ml of EGF for 5 or 10 minutes and the ERK activities were determined by Western blotting. (E-F) Cell motility was determined by wound healing assay in μ-Dish for NIH/3T3 (E) and CMT-93 (F) stable cell lines. (G) The kinetic information about the migration of CMT-93 stable cell lines was recorded using the x CELLigence RTCA-DP system. (H) Stable CMT-93 cell lines were seeded in 2 ml of medium containing 20% FBS with 0.35% agar at 5 × 103 cells/well and layered onto the base. The photographs were taken 2 weeks later. Images are representative of three independent experiments. (I) Stable CMT-93 cell lines (2 × 106) were injected subcutaneously into male BALB/c nude mice (n = 5) individually. The sizes of tumors were measured at day 14, 17 and 20 after injection and the tumors were weighed.