Fig. 6

Protein kinase DNA-PK drives PD-L1 T20T22 dual-site phosphorylation to mediate protein stabilization. a Endogenous co-immunoprecipitation was used to verify the binding of DNA-PK to PD-L1. b Exogenous co-immunoprecipitation was used to verify the binding of DNA-PK and PD-L1 in 293 T cells in the presence of hsa_circ_0136666. c Exogenous co-immunoprecipitation was used to verify the binding of DNAPK and PD-L1 in 293 T cells in the presence of miR-375. d Immunofluorescence was carried out to confirm the colocalization of DNA-PK and PD-L1. e Rescue experiment using phosphorylated acrylamide SDS-PAGE to detect phosphorylated PD-L1. f The PRKDC inhibitor NU-7441 decreased PD-L1 protein expression in a concentration dependent manner. g Detection of phosphorylated PD-L1 after single mutation of seven phosphosites. h Detection of phosphorylated PD-L1 after multiple mutations. i Detection of phosphorylated PD-L1 after double phosphorylation site mutation under MG-132 treatment. j Quantification of phospho-PD-L1 after mutation of dual phosphorylation sites, n = 3. k CHX experiment was used to verify the stability of PD-L1 protein. l Quantification plot of PD-L1 protein stability, n = 3. m Clinical data of 375 groups of CD274 and PRKDC genes analyzed by TCGA database. Data are presented as the mean ± SD, Student's t-test was used. **P < 0.01, ****P < 0.0001