SDS-PAGE Analysis of Protein Expression

Protein expression was examined by SDS-PAGE, as described previously [20 (link)]. Briefly, total intracellular proteins were extracted from the cells by freeze/thawing in lysis buffer containing protease inhibitors. Protein content was estimated according to Biorad protein assay (BIO-RAD). A total of 20 µg of proteins were visualized using the chemioluminescence detection system (Amersham biosciences; Little Chalfont, UK) after incubation with rabbit polyclonal primary antibodies against ANXA1 (1:10,000; Invitrogen; Carlsbad, CA, USA) and calreticulin (1:1000; Elabscience; Houston, TX, USA), with mouse monoclonal primary antibodies against TSG101 (1:1000; ThermoFisher Scientific; Waltham, MA, USA) and anti-β actin (mouse monoclonal; 1:1000; clone AC15; A5441, Sigma-Aldrich). The blots were exposed and analysed to Las4000 (GE Healthcare Life Sciences).