SPIN90 knockdown increases the levels of surface EGFR.

A. HeLa cells were transiently transfected with either siRNA-hH1zeo-G2 vector or siRNA-hH1zeo-G2-SPIN90 1731 (si-1731) for knockdown of SPIN90. (a) SPIN90 knockdown was verified by immunoblotting using an anti-SPIN90 antibody. (b) Graphs indicate the levels of TR-EGF localized at membrane in si-1731 transfected cells and non-transfected cells. (c) siRNA-hHazeo-G2-SPIN90 1731 transfected cells were visualized by green fluorescent proteins (green). An untransfected control cell is marked by a dotted line. B. (a) Control and SPIN90 knockdown (KD) HeLa cells were treated with 40 ng/ml Texas Red (TR)-EGF for 30 min at 4°C, followed by additional incubation for 0 or 10 min. Cells were fixed and stained with polyclonal anti-EGFR antibody and FITC secondary antibody. (b) The fluorescence intensity of membrane EGFR labeled with TR-EGF was quantified using Metamorph software. C. Cells were starved for 16 h and stained with monoclonal anti-EGFR (528) antibody for 30 min at 4°C. Cell lysates were incubated with protein A/G Sepharose beads for 2 h, and resolved using SDS-PAGE. (a) Surface EGFR was blotted with anti- EGFR antibody. (b) is the surface EGFR-quantified result of (a).