Fig. 7: YTHDF1 interacts with eIF5B and promotes the translation of PD-L1.

A Silver-stained gel revealed specific protein bands (highlighted with arrows) within the Flag-YTHDF1 immunoprecipitation sample, subsequently analyzed by LC–MS/MS. B Mass spectrometry identified eIF5B, which was precipitated from T24 cell lysates using a Flag antibody. C A schematic illustrates the full-length and truncated forms of eIF5B. D, E Co-immunoprecipitation assays identified the region of eIF5B interacting with YTHDF1. T24 cells transfected with various truncations underwent immunoprecipitation with an anti-Flag antibody targeting eIF5B, followed by immunoblotting using anti-Flag or anti-YTHDF1 antibodies. F Representative immunohistochemistry images of adjacent normal and tumor tissues from bladder cancer patients showing the correlation between YTHDF1 and eIF5B expression. G Correlation analysis of immunohistochemical scores for YTHDF1 and eIF5B in tumor tissues of bladder cancer patients.H Representative immunofluorescence images of tumor tissues from bladder cancer patients showing the correlation between endogenous YTHDF1 and eIF5B. I Alphafold 3 prediction of the binding between eIF5B protein and PD-L1 mRNA. J Western Blot analysis showing the expression of YTHDF1, eIF5B, and PD-L1 proteins after knocking down YTHDF1 in T24 and 5637 cells. K Western Blot analysis showing the expression of eIF5B and PD-L1 proteins after treating T24 and 5637 cells with different concentrations of methionine. (L-M) Western blot analysis of YTHDF1, eIF5B, and PD-L1 expression in T24 (L) and 5637 (M) cells with shYTHDF1 and shNC treatments, in the presence or absence of methionine and IFN-γ. N Representative histograms and bar graphs showing changes in PD-L1 expression in T24 cells after knocking down YTHDF1 and/or overexpressing eIF5B. **P < 0.01, ***P < 0.001, ****P < 0.0001. LC–MS/MS liquid chromatography-tandem mass spectrometry.