Fig. 6

US11 induces FcRn dislocation and ubiquitylation. a–d FcRn is ubiquitinated in the presence of US11 and MG132. HeLa^FcRn (a), HeLa^HFE (b), HeLa^{FcRn CT−/−} (c, lane 2), and HeLa^{FcRn 365A−/−} (c, lane 3) cells were transfected with or without US11 plasmids for 48 h, and cells were treated with proteasome inhibitor MG132 (50 μM) for 2 h, as indicated. HeLa^FcRn or HeLa^FcRn+US11 cells (d) were treated with CHX (100 μg/ml) and chased for the indicated time in the presence of MG132. Cell lysates (0.5 mg) were immunoprecipitated with mAb anti-FLAG for FcRn (a, c, d) or HFE (b). All immunoprecipitates or cell lysates (20 μg) as internal controls were subject to western blotting with corresponding Abs as indicated. e Fractionation of FcRn HC. HeLa^FcRn, HeLa^US11+FcRn cells were incubated in the presence or absence of 50 μM MG132 for 4 h. Cells were then homogenized and the homogenates were fractionated by centrifugation (see Methods). FcRn in the membrane pellet (M, lanes 1, 3, 5, 7) and supernatant (S, lanes 2, 4, 6, 8) fraction was digested by mock (top), Endo-H (middle), PNGase F (bottom) enzymes for 2 h at 37 °C, respectively. R: resistant; S: sensitive. After treatment all fractions were subject to western blotting with corresponding Abs as indicated