Figure 2. CCRP is ubiquitinated upon TCPOBOP treatment.

Various combinations of the following plasmids were transfected into HepG2 cells 18(cell density 5×10⁶ cells/10 cm plate): pcDNA3.1/V5-His-mCCRP (3 µg), pCR3-mCAR-FLAG (3 µg), and pCW7-Myc-Ub (3 µg). 24 hr after transfection, cells were treatment with DMSO (0.1% v/v), MG132 (5 µM in 0.1 1% DMSO, final concentrations) or MG132 (5 µM) in combination with TCPOBOP (250 nM dissolved in 0.1% DMSO, final concentrations). At the end of treatment, cells were harvested, cytosolic extracts prepared, and immunoprecipitation analysis was performed to detect ubiquitinated CCRP and CAR. Results shown are representative of three independent experiments. (A) Cells were transfected pcDNA3.1/V5-His-mCCRP (3 µg) and pCR3-mCAR-FLAG (3 µg) with or without pCW7-Myc-Ub (3 µg). Immunoprecipitation using anti-V5 antibodies or anti-IgG (as control) was performed, followed by immunoblotting analysis using anti-V5 and anti-Myc antibodies. (B) As a control for detecting ubiquitinated CCRP, extracts derived from cells cotransfected with empty vector or pcDNA3.1/V5-His-mCCRP and pCW7-Myv-Ub were subjected to immunoprecipation then immunoblotting analysis in (A). (C) Extracts were also subjected to imunoprecipitation analysis using anti-FLAG antibodies or anti-IgG (as control), followed by immunoblotting analysis using anti-FLAG and anti-Myc antibodies. (D) Immunblotting results of extracts used in the immunoprecipitation experiments, using anti-V5, anti-FLAG, and anti-Myc antibodies.