Fig. 5

TMEM52B suppression promotes phosphorylation and internalization of EGFR and downstream signaling. (A) Cells were transfected with shRNA specific to TMEM52B for 48 h and treated with EGF (10 ng/ml) for the indicated time prior to lysis for immunoblot analysis. Densitometric quantification was performed on the immunoblots; phosphorylated proteins were normalized against the corresponding total protein. (B) SW480sub cells were transfected with shRNA for 48 h and treated with EGF (100 ng/ml) for the indicated times. EGFR, EEA1, and DAPI were visualized; green for EEA1, red for EGFR, and blue for DAPI staining. Scale bar, 20 μm. Co-localization of EGFR and EEA1 was quantitated by calculating Pearson’s correlation coefficient using ImageJ software. Values represent mean ± SD. *P < 0.05. (C) Cells transfected with shRNA specific to TMEM52B for 42 h were treated with dynasore (160 μM) or DMSO as a vehicle control for 6 h and then treated with EGF (10 ng/ml) for 5 min prior to lysis for immunoblotting. (D, E) Cells were transfected with shRNA specific to TMEM52B for 48 h. Cell survival (D) and invasion (E) assays were performed as described in Fig. 2A and B in the presence of dynasore (5 μM or 10 μM) and EGF (10 ng/ml). Cell invasion was determined by calculating the cell-stained area relative to the total area using ImageJ software. All determinations were performed in three independent experiments. Values represent mean ± SD. *P < 0.05 compared with shControl + DMSO; ^§P < 0.05 compared with shTMEM52B + DMSO