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Fig. 3. Polyubiquitylated SCD1 accumulates in the presence of MG132. (A) CHO-K1 cells were transfected with pcDNA3.1 or the plasmid harboring SCD1-HA and grown for 48 hours. DMSO or 50 µM MG132 was then added to the cells and culture was continued for 5 hours. The cells were solubilized with buffer containing SDS (see Materials and Methods) and subjected to immunoprecipitation using anti-HA antibodies. The immunoprecipitates were analyzed by SDS-PAGE and subsequent immunoblotting with anti-polyubiquitin antibodies (upper panel) or anti-HA antibodies (lower panel). Lane 1 (IP control): immunoprecipitation without cell lysate. (B) NIH 3T3-L1 cells were grown in the differentiation cocktail for 10 days, DMSO or 50 µM MG132 was added, and culture continued for 5 hours. The cells were subjected to immunoprecipitation using anti-SCD1 C-terminal peptide antibodies or preimmune IgG and the immunoprecipitates were analyzed by SDS-PAGE and subsequent immunoblotting with anti-polyubiquitin (upper panels) or SCD1-C-peptide antibodies (lower panels). Lane 1 (IP control): immunoprecipitation without cell lysate. (C) CHO-K1 cells were transfected with the indicated plasmids and grown for 48 hours. MG132 (50 µM) was added to the cells and culture was continued for 5 hours. The cells were solubilized using SDS-buffer and subjected to immunoprecipitation using anti-Myc antibody. The immunoprecipitates were analyzed by SDS-PAGE and subsequent immunoblotting with anti-HA antibody. Magic Mark XP (Invitrogen) containing IgG-binding sequence-tagged proteins was used as molecular mass markers; detected by immunoblotting.
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