Fig. 5: Homodimerized intracellular domains regulate PD-L1 complex glycosylation.
From: Homodimerized cytoplasmic domain of PD-L1 regulates its complex glycosylation in living cells
a PD-L1 forms more homodimers in the Endoplasmic reticulum. HEK293T cells were transiently transfected with PD-L1-I247tag-Flag and pIRE4-Azi plasmids. 1 mM of Azi was added to the medium. Cells were treated with UV for 15 min. The ER, Golgi, and plasma membrane fractionation were isolated and separated on SDS-PAGE gels. Immunoblotting was performed using anti-Flag antibodies. b PD-L1△C homodimerized with WT PD-L1. Azi was genetically incorporated into V76 of PD-L1-Flag or PD-L1△C-Flag in HEK293T cells expressing PD-L1-HA. Cells were treated with UV for 20 min. Cell lysates were treated with PNGase F to remove the N-glycan. c, e PD-L1△C suppresses complex glycosylation of WT PD-L1. c, d WT PD-L1-HA and PD-L1△C-Flag plasmids were transfected into RKO KO PD-L1 Cells. c PD-L1△C affects the pattern of PD-L1 that showed on immunoblotting. The anti-PD-L1 antibodies (clone number E1L3N) could recognize the full-length PD-L1, but not the PD-L1△C. d Cell lysates for lane 3 and lane 4 were treated with PNGase F. Samples for lane 5 and lane 6 were treated with 10 µM tunicamycin (TM) for 24 h before cell collecting. e RKO KO PD-L1 cells were cotransfected with PD-L1-Flag and PD-L1△C-Flag plasmids. Cell lysates for lanes 4–6 were treated with Endo H. Lines and arrowheads colored blue indicate full-length PD-L1. Red arrowheads indicate the PD-L1△C proteins.
