Anti β actin mouse antibody

Total proteins were extracted from homogenized vessels using TRIzol as recommended by the manufacturer (Invitrogen; Thermo Fisher Scientific, Inc.). Protein extracts from two rats of each group were resuspended in 1% SDS supplemented with phosphatase and a protease inhibitor cocktail (New England Biolabs, Inc.). The soluble protein concentration was determined using a BCA Protein Assay kit (Thermo Fisher Scientific, Inc.). For western blotting analysis, 20 µg of total protein extract were resolved by SDS-PAGE on 12% gels (41 (link)). The proteins were transferred to nitrocellulose membranes, blocked for 1 h at room temperature in PBS containing 5% non-fat dry milk and 0.1% Tween-20, then incubated overnight at 4°C with the anti-ERp72 rabbit polyclonal antibody (1:1,000; Abcam; cat. no. ab82587) or anti-β-actin mouse antibody (1:1,000; BD Biosciences; cat. no. 612656) (41 (link)). The respective HRP-conjugated secondary antibodies (anti-rabbit IgG HRP-linked antibody cat. no. 7074 and anti-mouse IgG HRP-linked antibody cat. no. 7076; Cell Signaling Technology, Inc.) were used at a dilution of 1:1,000 for 2 h at room temperature for the detection step. The bands were detected using an Enhanced Chemiluminescence kit (Cytiva) and images were acquired using an LAS-1000 plus image analyzer with Image Reader LAS-1000 Lite software ver. 2.2 (Fujifilm Wako Pure Chemical Corporation).