Fig. 5

GSDME knockdown promotes the degradation of EGFR. A Western blot analysis of EGFR and GSDME levels in A549 and H1299 cells treated with 20 μg/mL cycloheximide (CHX) for 0 h, 6 h, 12 h and 24 h after transfection with siScr or siGSDME for 24 h. β-Actin was used as the loading control. B Western blot analysis of EGFR and GSDME levels in H157 cells treated with 15 μM E64D or 20 μM MG132 for 6 h after transfection with siScr or siGSDME for 24 h. GAPDH was used as the loading control. C EGFR ubiquitination was detected by a co-IP assay in GSDME-depleted H157 cells. H157 cells were transfected with pcDNA3.1-HA-EGFR, pcDNA3.1-HIS-Ub, siScr or siGSDME for 24 h and then treated with 15 μM E64D, 20 μM MG132 for 6 h and 100 ng/mL EGF for 15 min. A co-IP assay was performed with HA antibody, and the coeluted proteins were detected by Western blot analysis with HIS, HA and GSDME antibodies. The numbers shown above the bands were fold changes of band intensities relative to the control. The band intensities of HIS were quantified by Image J software and normalized to HA. The band intensity of HIS was relatively increased in GSDME-knockdown H157 cells. GAPDH was used as the loading control for the input. D Up: Immunofluorescence of EGFR (red fluorescence) and EEA1 (green fluorescence) in GSDME-depleted A549 cells. Scale bar, 10 μm. Down: quantification of the co-localization of EEA1 and EGFR. RNAi effency of A549 cells were measured by Western blot analysis. β-Actin was used as the loading control. E The interaction of EGFR and c-Cbl was detected by a co-IP assay in GSDME-depleted H1792 cells. Cells were transfected with pcDNA3.1-HA-EGFR, siScr or siGSDME for 24 h and then treated with 15 μM E64D, 20 μM MG132 for 6 h and 5 ng/mL EGF for 10 min. A Co-IP assay was performed with HA antibody, and the coeluted proteins were detected by Western blot analysis with c-Cbl, HA and GSDME antibodies. The numbers shown above the bands were fold changes of band intensities relative to the control. The band intensity of c-Cbl was quantified by Image J software and normalized to HA. c-Cbl level was relatively increased in GSDME knockdown H1792 cells. β-Actin was used as the loading control of the input. F Western blot analysis of phosphorylated EGFR (Y1045), EGFR and GSDME levels in A549 cells treated with 15 μM E64D or 20 μM MG132 for 6 h and 10 ng/mL EGF for 0 min, 10 min, 20 min, 40 min and 80 min after transfection with siScr or siGSDME for 24 h. G Western blot analysis of phosphorylated EGFR (Y1045), EGFR and GSDME levels in H157 cells treated with 15 μM E64D or 20 μM MG132 for 6 h and 10 ng/mL EGF for 0 min, 10 min, 20 min and 40 min after transfection with siScr or siGSDME for 24 h. β-Actin was used as the loading control