Ma5 13269

Cells were grown on glass slides in 24-well plates under standard conditions for 48 hr. Slides were then rinsed in PBS and fixed for 15 min in 4% PFA at 37°C. After washing with PBS, cells were permeabilized with PBS containing 0.2% Triton X-100% and 0.5% BSA for 5 min, and then blocked for 1 hr in PBS containing 0.5% BSA and 0.2% gelatine. Primary antibodies were added in blocking solution for 1 hr at room temperature in a humid chamber. The cells were rinsed in PBS three times before being incubated for 40 min in the dark with secondary antibodies and Phalloidin 568 (B3475 Thermo Scientific). After three washes with PBS, cells were stained for 5 min with PBS containing 0.1 µg/ml DAPI, followed by three washes with PBS. Glass slides were mounted with ProLong Gold Antifade Mountant (Thermo Scientific). The antibodies used for immunocytochemistry were: anti-CHORDC1 (HPA041040 Atlas Antibodies), anti-Tubulin (ab18251 abcam), anti-EGFR (MA5-13269 Thermo Scientific), anti-PDI (MA3-019 Thermo Scientific), Alexa Fluor 488 (A11034 Thermo Scientific), and Alexa Fluor 647 (A21236 Thermo Scientific). For all antibody stainings, at least three biological replicates were made.