Rabbit anti calr

Protein content was estimated by 1D-SDS-PAGE/SYPRO^® Ruby protein staining densitometry, as previously described173 (link). For immunoblotting (10 μg), membranes were probed with primary antibodies [mouse anti-TSG101 (BD Transduction Laboratories; 1:500), mouse anti-Alix (Cell Signaling Technology; 1:1000), rabbit anti-CD70 (Abcam; 1:1000), mouse anti-HSPG1 (Abcam; 1:200), rabbit anti-MSLN (Cell Signaling Technology; 1:1000), rabbit anti-FAT1 (GeneTex; 1:2000), mouse anti-CD81 (Santa Cruz Biotechnology; 1:1000), rabbit anti-CALR (Abcam; 1:1000) for 3 hr at room temperature (RT) in 50 mM Tris, 150 mM NaCl, 0.05% (v/v) Tween 20 (TTBS) followed by incubation with either IRDye 800 goat anti-mouse or goat IgG or IRDye 680 goat anti-rabbit IgG (1:15000, LI-COR Biosciences) for 1 hr at RT in TTBS. Immunoblots were visualized using the Odyssey Infrared Imaging System and Image Studio™ Software (v3.0, LI-COR Biosciences, Nebraska USA).

Total cellular proteins were extracted using RIPA buffer, and the cell lysates were centrifuged at 15,000 g at 4°C for 15 min. The protein concentration was measured using the BCA protein assay (Beyotime, China). Then, 30 µg protein was separated on a 10% SDS-PAGE, and the separated proteins were electro-transferred onto the 0.2-µm PVDF membranes. The membranes were then blocked in TBST containing 5% nonfat dry milk for 2 h at room temperature and hybridized with the primary antibody (rabbit anti-CALR, Abcam) overnight at 4°C. Then, the blots were probed with HRP-conjugated secondary antibodies and detected using ECL, photographed, and quantified using ImageJ software.