FIGURE 2.
Insig-2(L210A) is ubiquitinated by gp78. A, on day 0 Scap-deficient SRD-13A cells were set up at 3.5 × 105 cells/60-mm dish as described under “Experimental Procedures.” On day 2 each dish of cells was switched to medium A supplemented with 5% FCS and transfected with 0.2 μg of pEF1a-HA-ubiquitin together with one of the following plasmids: 0.1 μg pCMV-Insig1-T7, 0.3 μg pCMV-Insig2-T7, or 1.2 μg pCMV-Insig2(L210A)-T7. The total amount of DNA was adjusted to 1.4 μg/dish with pcDNA3 empty vector. After incubation for 16 h at 37 °C, the cells were incubated with 10 μm MG-132 for 30 min and harvested for preparation of detergent-solubilized cell lysates, which were subjected to immunoprecipitation with anti-T7 to precipitate Insigs. The immunoprecipitates (IP) were analyzed by immunoblot analysis (IB) with anti-HA (against ubiquitin) and anti-T7 (against Insigs). B, SRD-13A cells were set up and transfected with 0.1 μg of pCIneo-gp78 and one of the plasmids, 0.15 μg of pCMV-Insig1-T7, 0.7 μg of pCMV-Insig2-T7, and 2.1 μg of pCMV-Insig2(L210A)-T7, as described in A. The total amount of DNA was adjusted to 2.2 μg/dish with pcDNA3 empty vector. After incubation for 16 h at 37 °C, the cells were treated with 10 μm MG-132 for 2 h and then harvested, lysed, and immunoprecipitated with anti-T7 to precipitate Insigs. Pellets (representing 0.5 dish of cells) and supernatants (Sup; representing 0.05 dish of cells) were subjected to SDS/PAGE and immunoblot analysis with anti-T7 (against Insigs) and anti-gp78. C, on day 0 SV589 cells were set up at 2.0 × 105 cells/60-mm dish in medium C. On day 1, cells were transfected with 0.6 μg of pCMV-Insig2(L210A)-Myc. After incubation for 4 h at 37 °C, cells were transfected with 400 pmol/dish siRNA duplexes targeting green fluorescent protein or gp78. On day 3 cells were treated with 50 μm cycloheximide (CHX). After the indicated period of time, cells were harvested, and detergent lysates were subjected to SDS/PAGE followed by immunoblot analysis with anti-Myc (against Insig-2) and anti-gp78.