Fig. 6.

Increased efficiency of MHC II endocytosis in MARCH-I–overexpressing B cells. (A) Western blot of WCL and MHC II IPs of wild-type splenic B blasts retrovirally transduced to express MARCH-I (M1) Ub ligase. GFP(+)-transduced cells were sorted, with GFP(-) cells as control. MHC II was immunoprecipitated from cell lysates with anti-MHC II TIB120 antibody. IPs and WCLs were immunoblotted with anti-Ub antibody P4D1. Arrowhead approximates band migration of di-ubiquitinated MHC II. (B) Confocal microscopy of M1-expressing B blasts. Cells were bound to coverslips, fixed in PFA, and labeled with TIB120 (red) and lysosomal marker H2-M (blue). (C) FACS analysis of surface-bound MHC in unsorted M1-expressing B blasts. Cells were stained with PE-conjugated TIB120. (D) Confocal microscopy of surface MHC II uptake in M1-expressing B blasts. B blasts were incubated with TIB120 for 30 min at 37 °C for uptake, extensively washed, fixed, and stained to detect TIB120 (red) and H2-M (blue). (E) Quantification of TIB120 internalization as described in Fig. 3E for cells with low-, mid-, and high-intensity GFP. Data are expressed as mean and SEM. (F) Endocytosis of surface MHC II in M1-expressing B cells. Acid wash protocol on sorted GFP(+) (“M1”) and GFP(-) (“CTL”) cells as described in Fig. 1C.