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. 2009 Sep 7;186(5):685–692. doi: 10.1083/jcb.200906110

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© 2009 Stagg et al.

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Figure 2. — US2-dependent MHC I dislocation and ubiquitination are abolished after TRC8 depletion. (A) Cellular fractionation shows that TRC8 depletion rescues MHC I from the soluble to the membrane fraction. siRNA mock (Mk) or TRC8-depleted cells ± US2 were separated into membrane (M) and soluble (S) fractions. MHC I was immunoprecipitated (mAb HC10 or w6/32) from each fraction and visualized by immunoblot analysis (mAb 3B10.7). After TRC8 depletion, conformational MHC I is found in the membrane fraction. (B and C) TRC8 depletion prevents MHC I ubiquitination. (B) Detergent lysates from 20-min radiolabeled control (ctrl) and US2 cells were immunoprecipitated with the MHC I (w6/32) mAb, denatured, and reprecipitated with heavy chain (HC10)– or polyubiquitin (P4D1)-specific mAb. HC10 precipitates were confirmed as ubiquitinated MHC I species. (C) siRNA mock or TRC8-depleted control and US2 cells were treated as in B. Ubiquitinated MHC I species were lost after TRC8 depletion. (A–C) Molecular mass is indicated in kilodaltons. 1°, primary; 2°, secondary; HC, heavy chain; IP, immunoprecipitation.