Expression, phosphorylation, and subcellular distribution of c-Cbl in ICSBP+/+ and ICSBP−/− BMMs.
Cells were deprived of CSF-1 for 16 hours, harvested, and lysed in RIPA buffer. (A) Total cell extracts of 5 × 105 cells were subjected to western blotting with anti–c-Cbl antibody. Anti-Fyn antibody was used as control. (B) BMMs deprived of CSF-1 for 16 hours were left untreated or were stimulated for 4 minutes with 200 ng/mL CSF-1 at 37°C. Total cell extracts (1.5 mg) were used for immunoprecipitation with a c-Cbl antibody. The precipitates were subjected to western blot analyses with an antiphospho-tyrosine antibody (upper panels). After stripping the membrane was probed with an anti–c-Cbl antibody (lower panels). (C) BMMs continuously grown in the presence of 20 ng/mL CSF-1 were fractionated as described in “Materials and methods.” A quantity of 25 μg of membrane (Me) and cytosolic fraction (Cy) were subjected to western blotting with an anti–c-Cbl antibody (upper panel). Equal protein loading was controlled by the detection of p80 with the anti-ICSBP antiserum (lower panel).
