Tyrosine phosphorylation of total cellular lysates. Protein was generated from Ba/F3-mMPL, DA-1–TPO, L8057 cells, and megakaryocytes before (−) and after (+) TPO-stimulation for 15 minutes, as described in the Materials and Methods. Fifty micrograms of each sample was evaluated by Western blot and probed with a phosphotyrosine-specific antibody (P-TYR). The apparent decreased intensity of a 70-kD band in the last lane was an artifact due to incomplete transfer and was not seen in four similar experiments. The blot was stripped and reprobed with an MPL-specific antiserum (lower strip).