Figure 2. Molecular evaluation of the SHOC2 p.M173I variant.

A, Cos-LV1 cells were transiently transfected with full-length SHOC2-tRFP or SHOC2 (M173)-tRFP truncated mutants. Cells were serum-starved for up to 10 hours and treated with 0.2ng/ml EGF for indicated times at 37°C. The lysates were probed by immuno-blotting for EGFR, RAF-1, SHOC2, activated MEK1/2 (pMEK1/2), activated ERK1/2 (pERK1/2), total ERK1/2 (tERK1/2) and GAPDH (loading control). Dotted line shows area of the blot that was cropped to conserve space. B, Multiple blots from the experiments exemplified in A were analyzed. Bars represent the mean values (±S.E., n = 3) of phosphorylated ERK1/2 activity normalized to total ERK1/2 in arbitrary units (pERK/ERK), (a vs. b, c vs. d, P < 0.05, one-way ANOVA followed by a Student’s t-test was used to determine differences in phosphorylated ERK1/2). C, 293FT cells transiently co-transfected with expression vectors encoding GST-SHOC2 and HA-M-RAS. GST-SHOC2 were immunoprecipitated (IP) with glutathione sepharose beads, and the immunoprecipitates were probed with GST antibodies to detect SHOC2, RAF-1, PP1c and HA antibodies to detect M-RAS. Cell lysates were immunoblotted with anti-SHOC2, anti-RAF-1, anti-PP1c and anti-HA antibody. D, Cos-LV1 cells transiently expressing SHOC2 (WT)-tRFP and SHOC2(M173I)-tRFP were serum-starved for up to 10 hours and treated with 0.2ng/ml EGF for indicated times at 37°C. The lysates were probed by immuno-blotting for SHOC2, phospho-(S338) RAF-1, activated ERK1/2 (pERK1/2), and GAPDH (loading control). Results in each panel are representative of three independent experiments. E, Multiple blots from the experiments exemplified in D were analyzed. Bars represent the mean values (±S.E., n = 3) of phosphorylated RAF-1 (7 min of EGF treatment) normalized to GAPDH in arbitrary units (pRAF-1/GAPDH) (a vs. b, P = 0.029).