Ecl advance western blotting detection reagents

Western blotting was performed as described previously (Yu et al., 2019 (link)). In brief, protein samples were separated in 10–15% SDS-PAGE acrylamide gels, transferred onto PVDF membranes (Millipore, 0.22μm and blocked with 5% skim milk for 2h). The blots were then incubated at 4°C overnight with the following primary antibodies: Claudin-5 (1:1,000; Invitrogen, 34–1,600), LC3 (1:1,000; ProteinTech, 14600-1-AP), Caveolin-1 (1:200; Santa Cruz Biotechnology, sc-894), HIF-1a (1:1000; ProteinTech, 20960-1-AP), iNOS (1:1,000; ProteinTech, 18985-1-AP), LAMP-1 (1:1000; ProteinTech, 21997-1-AP), ATP1A1 (1:1,000, ProteinTech, 14418-1-AP), or β-actin (1:1,000; Servicebio, GB12001). Then, the blots were incubated at room temperature for 2h with HRP conjugated anti-rabbit or anti-mouse IgG (H+L; 1:1,000; Servicebio) secondary antibodies. Western blot bands were analyzed by adding ECL advance Western blotting detection reagents (Thermo, 34,580) and imaged using a SmartChemi-500 imaging system (Sage Creation Science, China). The immunoblot bands were quantitatively analyzed by ImageJ software.

Isolated mouse cardiomyocytes or NRCMs were lysed with a lysis buffer supplemented with a protease inhibitor cocktail and PMSF to prepare the total protein extracts. The protein samples were separated by SDS-PAGE and transferred into nitrocellulose membranes, which were blocked in 5% non-fat milk for 1 h at room temperature followed by incubating with primary antibodies overnight at 4 °C. Then, the samples were incubated with HRP-conjugated anti-mouse or anti-rabbit secondary antibodies (7076S; 7074S; Cell Signaling Technology, Denvers, MA, USA) for 1 h at room temperature. Immunoblot signals were incubated with ECL Advance Western blotting detection reagents (Thermo Scientific, Waltham, MA, USA, 34577) and detected by Tanon (6100C). All the protein levels were normalized by GAPDH. The following primary antibodies were used in the present study: anti-RBFox2 (Bethyl, Montgomery, TX, USA, A300-864A); anti-Jph2 (SantaCruz, Dallas, TX, USA, sc-377086); anti-Dorsha (SantaCruz, Dallas, TX, USA sc-393591); anti-Dicer (SantaCruz, Dallas, TX, USA sc-136979); and anti-GAPDH (FuDe, Wuhan, China, BM3874).