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. 2010 Dec 15;85(5):2288–2295. doi: 10.1128/JVI.01961-10

FIG. 4.

FIG. 4.

Palmitoylation of MIR2 is necessary for MIR2-mediated ubiquitination of MHC-I. (A) HeLa cells or HeLa cells stably transduced with B7.2 were transfected with FLAG-tagged MIR2, FLAG-tagged MIR2-C146F, or vector. MHC-I or B7.2 was immunoprecipitated (IP) from cellular lysates using an anti-MHC-I or anti-B7.2 antibody, and the ubiquitination status of MHC-I (top left panel) or B7.2 (top right panel) was analyzed by Western blot analysis using an antiubiquitin antibody. FLAG-MIR2 and FLAG-MIR2-C146F were also immunoprecipitated from cellular lysates using an anti-FLAG antibody (M2 Sigma) and analyzed by Western blot analysis to verify equal expression levels (bottom panels). (B) HeLa cells stably transduced with FLAG-tagged MIR2 or FLAG-tagged MIR-C146F were lysed in co-IP lysis buffer. FLAG-MIR2 and FLAG-MIR2-C146F were immunoprecipitated from cellular lysates using an anti-FLAG antibody, and the levels of coimmunoprecipitating MHC-I were determined using the HC10 antibody (top panel). FLAG-MIR2 or FLAG-MIR2-C146F levels were analyzed by Western blot analysis using an anti-FLAG antibody (middle panel). As a control, levels of MHC-I in lysate samples were determined using the HC10 antibody (bottom panel).