FIGURE 8.
Ubiquitination of Insig-1 is not required for its interaction with p97. On day 0 Scap-deficient SRD-13A cells were set up at 6.0 × 105 cells/60-mm dish as described under “Experimental Procedures.” On day 1 each dish of cells was transfected with 0.3 μg pCMV-Myc-Ubxd8, pCMV-FLAG-Insig1, or pCMV-FLAG-Insig1(K156R/K158R) as indicated. The total amount of transfected DNA was adjusted to 1.3 μg/dish with pcDNA3 empty vector. After incubation for 6 h each dish of cells was switched to medium A supplemented with 5% delipidated FCS. On day 2 the cells were treated for 6 h with or without 100 μm sodium arachidonate-albumin in the presence of 10 μm MG-132. The cells were then harvested and lysed with detergent, and Insig-1 was immunoprecipitated with anti-FLAG-agarose beads. Pellets (representing 0.1 dish of cells) and supernatants (representing 0.01 dish) were subjected to SDS-PAGE followed by immunoblot analysis with anti-Myc (against Ubxd8), anti-FLAG (against Insig-1), and anti-p97. IP, immunoprecipitation; IB, immunoblot. This experiment was repeated two more times with similar results. To observe arachidonate-regulated binding between Insig-1 and p97, the amount of transfected plasmids had to be adjusted to prevent excessive overexpression of Insig-1.