Expression, autophosphorylation, and ubiquitination of CSF-1R in BMMs from ICSBP+/+ and ICSBP−/− mice.
(A) Total cell extracts from 5 × 105 BMMs cultured in the presence of 20 ng/mL CSF-1 (as described in “Materials and methods”) were loaded per lane and probed with an anti–CSF-1R antiserum by western blotting (upper panel). Equal loading of the lanes was confirmed by detection of an unspecific band, p80, with the ICSBP antibody (lower panel). (B) BMMs were deprived of CSF-1 for 16 hours, followed by stimulation with 100 ng/mL CSF-1 at 37°C for the indicated times. After incubation, cells were lysed and immunoprecipitated with an anti–CSF-1R antiserum. Immuncomplexes were analyzed by western blotting either with antiphosphotyrosine antibody (upper panel), antiubiquitin antibody (middle panel), or anti–CSF-1R antiserum (lower panel). In the CSF-1 western blot the position of the band corresponding to the 150-kd isoform of CSF-1 is indicated as a loading control.
