Cbl-b TKB and RF domains are required for Cbl-b-induced degradation and ubiquitination of KIT. The 293 cells were transfected with pCDNA3.c-KIT/pCEFL, pCDNA3/pCEFL.Cbl-b, or pCDNA3.c-KIT with different HA-tagged Cbl-b constructs in their expression vector (pCEFL) as indicated. Cells were starved and stimulated with or without SCF (100 ng/mL) for 10 minutes as in Figure 1. pCDNA3.c-KIT was cotransfected with a plasmid encoding Myc-tagged ubiquitin to facilitate detection of ubiquitylated KIT in panel B. (A) Cbl-b TKB and RF domains are required for Cbl-b-induced degradation of KIT. Cell lysate was immunoprecipitated with either α-KIT or α-HA antibody and probed with anti-KIT or α-HA antibody to show their protein levels. (B) Cbl-b TKB and RF domains are required for Cbl-b-induced 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 anti-KIT antibody.