Posttranscriptional regulation of c-Cbl.

(A) Unchanged levels of c-Cbl mRNA in BMMs from ICSBP^+/+and ICSBP^−/− mice. Isolation of RNA and northern blot analysis was performed as described in “Materials and methods.” The positions of the 10.5-kb c-Cbl transcript, 28S rRNA, and β-actin mRNA are indicated. (B-D) Effects of protease inhibitors on the expression of c-Cbl. (B) ICSBP^+/+ and ICSBP^−/− BMMs were grown in the presence of 20 ng/mL CSF-1 and treated with the protease inhibitor LLnL (25 μM) for 6 hours. Total cell extracts were analyzed by SDS-PAGE and western blotting with the anti–c-cbl antibody. Extracts from 5 × 10⁵ cells were used per lane. Equal loading of the lanes was confirmed by detection of p80 and ICSBP with the anti-ICSBP antiserum. (C) ICSBP^+/+ BMMs were treated with different protease inhibitors (25 μM) for 6 hours. Western blot analysis of the total cell extracts was performed with the antibodies as indicated. Lane 1: cathepsin B inhibitor (Z-Phe-Ala-CH₂F); lane 2: calpain inhibitor (Mu-Val-HPh-CH₂F); lane 3: lactacystin; lane 4: control (dimethyl sulfoxide [DMSO] solvent). Equal loading of the lanes was confirmed by detection of ICSBP. (D) ICSBP^+/+ BMMs were treated as in B and subcellular fractionation was performed as described in “Materials and methods.” Protein fractions (50 μg) were analyzed by SDS-PAGE and western blotting with the anti–c-Cbl antibody. Equal loading of the lanes was confirmed with detection of p80 and ICSBP by the anti-ICSBP antiserum.