Cbl-b and c-CBL induce the ubiquitination and degradation of KIT. The 293 cells were transfected with c-KIT expression plasmid along with expression vector (pCEFL; Vect) or different HA tagged Cbl-b or c-Cbl constructs. c-KIT was cotransfected together with a plasmid encoding Myc-tagged ubiquitin to facilitate detection of ubiquitylated KIT in panels B and C. Twenty hours later, cells were starved and stimulated with or without SCF as described in “Materials and methods.” (A) Cbl-b and c-Cbl induce the degradation of KIT. Whole-cell lysates were separated on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gel and immunoblotted (IB) for phosphotyrosine (α-pTyr), KIT (α-KIT), HA (α-HA), Cbl-b (α-Cbl-b), Cbl (α-Cbl), and p85 subunit of phosphatidyl-inositol 3-kinase antibodies (α-p85) as indicated. (B) Cbl-b induces the ubiquitination of KIT. Cell lysates were subjected to immunoprecipitation (IP) with anti-KIT antibody followed by blotting with anti-myc (to detect ubiquitylated KIT) antibody, stripped, and reprobed with KIT antibody. (C) Lactacystin, a proteasome inhibitor, blocks the degradation of ubiquitylated KIT induced by SCF/KIT. Cell lysates were subjected to IP and blotted with anti-myc antibody as in panel B. The membrane was subsequently stripped and probed with α-pTyr and α-KIT. In panels B and C, Ub indicates ubiquitin.