Ma3 019

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.

Cells grown on coverslips were fixed with 4% paraformaldehyde in PBS for 15 min at room temperature and permeabilized with 0.1% Triton X-100 in PBS for 20 min at room temperature. For immunostaining, the cells were incubated with various primary antibodies at 37 °C for 1 h, washed three times with PBS and incubated with appropriate secondary antibody, anti-rabbit IgG conjugated with Dylight405 (711-475-152, 1:1,000, Jackson ImmunoResearch, West Grove, PA) or anti-mouse conjugated with Alexa488 (715-545-150, 1:1,000, Jackson ImmunoResearch) at 37 °C for 1 h. The coverslips were washed three times with PBS and incubated with anti HLA-DP monoclonal antibody conjugated with DyLight594 (H1584, 1:200, Leinco Technologies) at 37 °C for 1 h, washed three times with PBS and mounted on a slide with antifade medium (Vectashield, Vector Labs, Burlingame, CA). Fluorescence images were captured using a confocal microscope (Zeiss LSM700). The primary antibodies utilized were as follows: rabbit anti-Ii polyclonal antibody (sc-20082, 1:200, Santa Cruz Biotechnology), mouse anti-Ii monoclonal antibody (sc-6262, 1:200, Santa Cruz Biotechnology), mouse anti-EEA1 monoclonal antibody (610457, 1:250, BD Biosciences), mouse anti-LAMP1 monoclonal antibody (sc-20011, 1:100, Santa Cruz Biotechnology) and mouse anti-PDI monoclonal antibody (MA3-019, 1:500, Thermo Scientific).