Anti beta actin

The protein concentration of each fraction was measured by a Bradford protein assay. An amount of 5 µg protein of each fraction was separated on 10% polyacrylamide gels at 40 mA per gel for 2 h and were blotted onto nitrocellulose membranes by semi-dry Western blot at 180 mA per membrane for 1 h. Membranes were blocked in 5% skim milk powder in TBS-T, incubated with the indicated primary antibody solution (in TBS-T supplemented with 5% BSA and 0.1% sodium azide) at 4 °C overnight followed by incubation with corresponding HRP-conjugated secondary antibody solution (in 3% BSA in TBS-T) at RT for 1 h. Proteins were visualized with ECL Detection Reagents (GE Healthcare, Chicago, IL, USA). The antibodies used were anti-beta Actin (MP, Irvine, CA, USA), anti-Tapasin, anti-beta2-Microglobulin, anti-ERp57, anti-Calreticulin (all Abcam, Cambridge, UK), anti-Yes (BD Bioscience), anti-Flotillin1, anti-Caveolin1 (Sigma-Aldrich, Seelze, Germany), anti-TAP1 (Novus Biologicals, Cambridge, UK), anti-TAP2 (MBL, Woburn, MA, USA), HRP-conjugated goat anti mouse and HRP-conjugated goat anti-rabbit (Thermo Scientific, Bonn, Germany).