Fig. 5. Overexpression of N4WBP5A inhibits receptor-mediated endocytosis. HeLa cells untransfected (A-C) or transfected with N4WBP5A-myc (D-L) were serum starved, cultured with EGF-Alexa Fluor 488 (200 ng/ml, green) for 1 hour at 4°C and EGF uptake was performed at 37°C for up to 25 minutes. Surface-bound EGF-Alexa fluor 488 was stripped from the cells, cells were fixed and permeabilized and immunohistochemistry performed with an anti-myc antibody and anti-mouse Alexa Fluor 568 secondary antibody (red). (A) EGF uptake for 5 minutes; (B) 10 minutes; (C) 20 minutes. (D-F) EGF uptake for 15 minutes. (G-I) EGF uptake for 20 minutes. (J-L) EGF uptake for 25 minutes. (A-C,D,G,J) EGF localization (green, transfected cells outlined). (E,H,K) Transfected cells expressing N4WBP5A (red) and merged images (F,I,L) showing transfected cells and endocytosed EGF Alexa Fluor 488. Bar, 10 µm.