Fig. 3.

Expression and endocytosis of MHC II–Ub fusion proteins in splenic B blasts differ depending on Ub extension. (A) Western blots of MHC II IPs and WCLs of MHC II β-chain^−/− splenic B blasts retrovirally transduced to express MHC II–Ub fusion constructs. MHC II was immunoprecipitated from cell lysates with anti-MHC II antibody Y3P. IPs and WCLs were immunoblotted with anti-Ub antibody (Biomol). Arrowhead approximates band migration of di-ubiquitinated MHC II. (B) Confocal microscopy of transduced B blasts. Cells were bound to coverslips, fixed in PFA, and labeled with TIB120 (red) and H2-M (blue). (C) MFI of surface-bound MHC II in transduced B blasts. Data are expressed as mean and SEM. (D) Confocal microscopy of surface MHC II uptake. Transduced 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 MHC II antibody internalization. The cell interior and exterior were identified on z axis sections at 2.0 ⌈m above the coverslip, and MHC II MFI was quantified for each region. Relative intensity of interior to exterior regions is shown as the mean and SEM.