Fig. 5: DNAJC5 enhances the endocytosis and recycle of EGFR.

A Immunofluorescence analysis was performed on serum-starved overnight A549 cells overexpressing DNAJC5 or control A549 cells, with or without treatment with EGF (10 ng/ml) for 15 minutes. Co-localization of EGFR and EEA1 was examined. B The degree of colocalization between EGFR and EEA1 was quantified using Pearson’s coefficient. C Immunofluorescence analysis was conducted on serum-starved overnight H1299 cells with knockdown of DNAJC5 or control cells, with or without treatment with EGF (10 ng/ml) for 15 minutes. Co-localization of EGFR and EEA1 was examined. D The degree of colocalization between EGFR and EEA1 was quantified using Pearson’s coefficient. E, F Overexpression of DNAJC5 promoted the endocytosis of EGFR, while knockdown of DNAJC5 suppressed it. To assess internalized EGFR levels, Sulfo-NHS-SS-biotin labeling was performed on ice followed by stimulation with EGF at 37°C for various durations. Cell lysates were immunoprecipitated and subjected to western blotting. Image J software was used to analyze the gray value corresponding to internalized EGFR. G Overexpression of DNAJC5 facilitated the recycling process of EGFR. In A549 cells, Sulfo-NHS-SS-biotin labeling on ice followed by stimulation with EGF at 37 °C for 15 minutes allowed surface biotin cleavage by MesNa. Subsequent incubation with EGF at 37 °C for different time periods induced the recycling process in which internalized EGFR returned to the cell surface. Cell lysates were immunoprecipitated and subjected to western blotting. Image J software was used to analyze the gray value corresponding to internalized EGFR.