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. 2022 Aug 30;5:887. doi: 10.1038/s42003-022-03845-4

Fig. 1. PD-L1 homodimerized in living cells.

Fig. 1

a Schematic diagram of genetically incorporated photocross-linking UAAs covalent capture PD-L1 homodimer. UAAs incorporated positions were referred with X or Y. PD-L1-X/YAzi-Flag, and WT PD-L1-HA were coexpressed in living cells. The anti-HA positive cross-linking band (colored red) is the complex of PD-L1-HA captured by PD-L1-XAzi-Flag. Red stars indicated the incorporated UAAs. Green curves indicated the HA tag and light blue curves indicated the Flag tag. b Structural formula of the 4-Azido-l-phenylalanine (Azi). c, d PD-L1-I247Azi captures homodimer dependent on the UAA incorporation and UV treatment. c Cells expressing PD-L1-I247Azi capture cross-linking bands in correspondence to PD-L1 homodimer. HEK293T cells were transiently transfected with PD-L1-I247tag-Flag and pIRE4-Azi plasmids. 1 mM of Azi was added to the medium. Cell lysates were separated on SDS-PAGE gels. Immunoblotting was performed using anti-Flag antibodies. d HEK293T cells were cotransfected with PD-L1-I247tag-Flag, pIRE4-Azi and PD-L1-HA plasmids. The quantity of PD-L1-I247tag-Flag plasmid increased from lane 4 to lane 6. All samples were treated with PNGase F to remove the N-glycan.