Kinase-mediated regulation of c-Cbl in CML cells. (A) K562 (left) and K562R (right) cells were electroporated with the indicated siRNA or left untreated (−) for 48 hours before cell lysates were analyzed for total tyrosine phosphorylation (top) or c-Cbl protein levels (bottom). Molecular mass marker migration is shown on the left, and specific target phosphoproteins are depicted on the right. (B) Cos-7 cells were transfected with cbl, bcr-abl, or lyn expression vectors alone or in combination (as indicated), and 24 hours later cells were lysed and changes in pY744-c-Cbl levels were monitored by phospho–site-specific immunoblotting. The membrane was stripped and reprobed for c-Cbl, BCR-ABL, and Lyn protein levels. Actin blotting was used as a protein loading control. (C) K562 cells were electroporated with a GFP-IRES Lyn expression vector (pMX-Lyn) or empty vector (pMX). After 7 days, cells were flow sorted for GFP positivity and equal numbers of positive cells were compared with untransfected K562 and K562R cells for Lyn, c-Cbl, and actin protein levels. (D) K562R cells were electroporated with control or c-Cbl–specific siRNA, and after 48 hours cell lysates were examined for c-Cbl, Lyn, and actin (as a protein loading control). Both a short and long exposure for Lyn detection is shown.