Fig. 5

B4GALT1 interacts with PD-L1 and enhances PD-L1 protein stability by mediating its glycosylation, thus promoting immune escape in early-stage LUAD. A The differential expression of immune checkpoint proteins in tumours with high and low B4GALT1 expression (data retrieved from TCGA-mRNA database). B, C A549 cells were transfected with si-B4GALT1 or pcDNA-B4GALT1, and PD-L1 expression was analysed by qRT‒PCR and western blotting. D A549 cells cocultured with activated CD8 + T cells for 48 h were subjected to crystal violet staining and quantified at 570 nm. The ratio of A549 cells to T cells was 1:3. E Western blot analysis of PD-L1 expression in A549 cells with or without B4GALT1 knockdown or overexpression followed by treatment with CHX. F Coimmunoprecipitation experiments indicated that B4GALT1 interacted with PD-L1 in both A549 cell lines. G Coomassie staining of GST and GST-B4GALT1 proteins (left). Detection of PD-L1 bound to GST-B4GALT1 or GST in a GST pull-down assay (right). H Western blot analysis of PD-L1 levels in B4GALT1 knockdown cells, B4GALT1-overexpressing cells, and tunicamycin (TM)-treated cells. I Western blot analysis of Flag-tagged WT or mutated PD-L1 expression levels in B4GALT1-overexpressing A549 cells followed by treatment with CHX. *P < 0.05, **P < 0.01, n.s., not significant