Fig. 3.

eEF1A1 and eEF1A2 form complexes with Shp2. (A) Complexes of His-tagged eEF1A1/eEF1A2 and Shp2 were precipitated from HEK293 stable cell lines using NiNTA sepharose. NiNTA sepharose beads were washed extensively and complexes were eluted by boiling in Laemmli sample buffer. Eluted proteins were resolved by SDS-PAGE and visualized by immunoblotting with anti-Shp2 (upper panel) or anti-His (lower panel) antibodies. NiNTA sepharose beads incubated with lysate of GFP overexpressing stable cell line were used as a control of non-specific binding of Shp2. TCL: total cell lysate. (B) Complexes were immunoprecipitated with anti-eEF1A antibodies from HEK293 cells treated under different conditions (growing, serum starved and serum starved followed by 30 min 10% serum stimulation). Protein A sepharose beads were washed extensively and the complexes were eluted by boiling in Laemmli sample buffer. Eluted proteins were resolved by SDS-PAGE and visualized by immunoblotting with anti-Shp2 antibodies. Protein A sepharose beads alone incubated with the same cell lysates were used as a control of Shp2 non-specific binding. Total cell lysates (TCL) of HEK293 cells were immunobloted with phosphospecific antibodies to phosphorylated Ser240/Thr244 in ribosomal protein S6 and β-actin to control the efficiency of treatment conditions and total protein amount used for immunoprecipitation in each sample, respectively. (C) GST or Shp2 GST-SH2 fusion proteins were coupled with glutathione-sepharose beads and incubated with phosphatase treated (10 U CIAP per 350 μg of total cell lysate) or untreated total cell lysates of stable cell lines overexpressing His-tagged eEF1A1 or eEF1A2 proteins. The beads were washed extensively and the proteins were eluted by boiling in Laemmli sample buffer. Eluted proteins were resolved by SDS-PAGE and visualized by immunoblotting with anti-His antibodies (upper panel). Amount of GST or Shp2 GST-SH2 proteins in each probe was controlled by immunoblotting with anti-GST antibodies (lower panel).