Anti pdi rl90

9×10⁴ COS7 cells were plated on coverslips. After 24 hours, cells were transfected and cultured for 24–48 hours. Cells were fixed with 3.7% formaldehyde for 1 hour at room temperature, permeabilized with 0.1% Triton X-100 for 5 minutes, and blocked with 1% BSA for 5 minutes. Cells were incubated with primary antibodies at 37C for 1–2 hours: anti-FLAG-M2 antibody (Sigma Aldrich #F1804), anti-phospho-HER3 Tyr1289 (21D3 – Cell Signaling), anti-MET (D1C2 – Cell Signaling), anti-phospho-MET Tyr1234/Y1235 (Cell Signaling), anti-phospho-EGF-Receptor Tyr1068 (Cell Signaling), anti-Golgin97 (A-21270 – Molecular Probes), anti-PDI (RL90 - Thermo Scientific), or anti-EEA1 (BD Biosciences - 610456), anti-HER3 (D22C5 XP – Cell Signaling). Cells were washed and incubated with fluorescently tagged anti-rabbit or anti-mouse secondary antibodies (Goat Anti-Mouse Alexa-fluor-568 cat#A11031; Alexa-Fluor-488 donkey anti-rabbit cat# A21206 – Life technologies). Images were taken at 60x magnification using a Nikon widefield epifluorescent microscope (Fig. 3a, b, 5a-d, 7b), or at 60x using a Nikon spinning disc confocal microscope Confocal images shown were taken as a stack of 7 slices in the Z-plane and processed as a Z-max projection (Supplementary Fig. 4).