![Fig. 5. 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.](https://ash.silverchair-cdn.com/ash/content_public/journal/blood/99/9/10.1182_blood.v99.9.3213/6/h80922464005.jpeg?Expires=1735556393&Signature=qHwWmGnbRSf8pgiKcihkZNBhJ230TbMAgH4c~4fJz0KxtbLBB5sM6O8OrJv7ZVAAH3lK7Smn1dStVv7iJ-kcNgmss5klrOjz9z5sNg1pBAPNW3qILpvhTVQLaSql6Bpq0Yyrt~yFkad2FWrpZj0~bM6MOfReMS5rOt5-sVrn7cJfSKYtdf4G9-g6zwImgKeJC1W5RAs0ShDLRyqTejdKcp5tFIu2CdtmBsWZtDBvwoQTcbgTvQA54Pnq3cZtklAweGzum2CQwiXNZPS45LImesGtetOA49sEg49dHzg1~n-nfp4aDBJ~ZbY7ZNFwrGyMBRxbBILZSLyDobkcCWy1AA__&Key-Pair-Id=APKAIE5G5CRDK6RD3PGA)
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.
