Figure 4
RPT3 deficiency is associated with increased 20S protease activity but decreased degradation of ubiquitinated substrates. (a) 30 μg of AT2 cell lysates were resolved by native gel electrophoresis followed by immunoblotting for 20S α subunits (1, 2, 3, 5, 6, 7) and 19S subunit RPT5, and overlay assay with Suc-LLVY-AMC substrate. 0.5 μg of human 26S proteasome and bovine 20S proteasome were used as controls. Addition of SDS stimulates 20S peptidase activity. Gel was stained post transfer with imperial blue to assess protein transfer. (b) 20 μg of AT2 cell lysates were separated by SDS-PAGE followed by immunoblotting for polyubiquitin and K-48 linked polyubiquitin. WT AT2 cells cultured on 100% Cultrex were treated with 10 nM Bortezomib (BZ) for 48 hours. Cultrex was dissolved using Cell Recovery Solution and cells were collected in 2X sample buffer containing BME for Western blotting. RPT3F/F - SftpcWT/CreER:RPT3F/F mice without tamoxifen treatment. Full-length blot for Actin is presented in Supplementary Fig. 9.
