MAPK phosphorylation is not fully dependent on Shc phosphorylation. Cell lysates were prepared either before (−) or after (+) stimulation with exogenous TPO (14 ng/mL for 10 minutes). Parental BAF3 cells were used as well as clones engineered to express the full-length murine Mpl receptor (mMpl) or mutated receptors, which were truncated after either 111 or 69 cytoplasmic amino acids (T-111 and T-69, respectively).11 (A) One hundred micrograms of each lysate was evaluated by Western blotting and probed to detect double-phosphorylated ERK1 and ERK2. The blot was stripped and reprobed with ERK2 antibody to confirm equal loading in all lanes. (B) Shc was immunoprecipitated from 1 mg of each cell lysate. The immunoprecipitated protein was evaluated by Western blotting and probed with a phosphotyrosine-specific antibody (4G10). The blot was stripped and reprobed to verify the presence of Shc in all lanes.