Fig. 2: Hsc70 induces PD-L1 degradation via eMI.

A, B MCF-7 cells were transfected with siRNAs of TSG101 for 36 h, transfected with or without Hsc70-Flag for another 24 h. Cell lysates were immunoblotted with indicated antibodies (A), fluorescence of PD-L1 on the surface of cell membrane was analyzed by flow cytometry (B). C, D MCF-7 cells were transfected with siRNAs of VPS4 for 36 h, transfected with or without Hsc70-Flag for another 24 h. Cell lysates were immunoblotted with indicated antibodies (C), fluorescence of PD-L1 on the surface of cell membrane was analyzed by flow cytometry (D). E, F MCF-7 cells were transfected with Flag or Hsc70-Flag for 12 h, treated with or without U18666A (5 μg/mL) for another 12 h. PD-L1 levels were detected by western blotting (E), fluorescence of PD-L1 on the surface of cell membrane was analyzed by flow cytometry (F). G, H MCF-7 cells were transfected with Hsc70-WT-Flag or Hsc70-3KA-Flag plasmids for 24 h. PD-L1 protein levels were detected by western blotting (G), fluorescence of PD-L1 on the surface of cell membrane was analyzed by flow cytometry (H). I–K MCF-7 cells were transfected with Flag, Hsc70-WT-Flag or Hsc70-3KA-Flag for 8 h, the co-localization between RAB7A and PD-L1 (I), the co-localization between LAMP1 and PD-L1 (J), the co-localization between RAB11 and PD-L1 (K) were done using immunofluorescence and confocal microscopy. Scale bar, 5 μm. The intensity profiles along the yellow line are plotted in the middle panels, with the colocalizing sites marked by white. For (B, D, F, H), MCF-7 cells were seeded in 48-well plate with 6 replicates per group and subjected to the corresponding treatment, repeated independently three times and similar results were obtained. Data shown in (A), (C), (E), (G), and (I–K) were repeated independently three times and similar results were obtained. The numbers under blots represent the value (the ratio to Tubulin) of grayscale quantification. Source data are provided as a Source data file.