Dylight 800 4xpeg conjugated goat anti rabbit igg h l

To obtain total protein extracts, cultured cells or frozen cardiac tissues were lysed at 4 °C in radioimmunoprecipitation (RIPA) buffer with protease and phosphatase inhibitor cocktail (Cat#P1050, Beyotime Biotechnology, China). Tissue homogenates or cell lysates were clarified by centrifugation at 14,000 g for 15 min at 4 °C and the protein concentration was determined using the bicinchoninic acid method (Cat#P0010S, Beyotime Biotechnology, China). A total of 20 μg protein per sample was size-fractionated by sodium dodecyl sulphate polyacrylamide gel electrophoresis and transferred onto Immobilon polyvinylidene difluoride membranes (Millipore, USA). The membranes were blocked for 2 h with 5 % DifcoTM Skim Milk (Cat#232100, BD Biosciences, USA) and then probed overnight at 4 °C with primary antibodies (Table S1), followed by a 1:30,000 dilution of Dylight™ 800 4XPEG-conjugated goat anti-rabbit IgG (H + L) or goat anti-mouse IgG (H + L) (Cell Signaling Technology, USA) for an hour. The results were visualized and analyzed by Odyssey Infrared Imaging System.

To obtain total protein extracts, cultured cells or frozen cardiac tissues were lysed at 4 °C in radioimmunoprecipitation (RIPA) buffer with cocktail protease inhibitor (Beyotime Biotechnology, China). Tissue homogenates or cell lysates were clarified by centrifugation at 12,000 rpm for 15 min at 4 °C and the supernatant protein concentration was determined using the bicinchoninic acid method (Beyotime Biotechnology, China). Proteins (20 μg) were size-fractionated by sodium dodecyl sulphate polyacrylamide gel electrophoresis and transferred onto Immobilon polyvinylidene difluoride membranes. The membranes were blocked for 2 h with 5% DifcoTM Skim Milk (BD Biosciences, USA) and then probed overnight at 4 °C with primary antibodies (Tbl.S3), followed by the secondary antibody for an hour with a 1:30,000 dilution of Dylight™ 800 4XPEG-conjugated goat anti-rabbit IgG (H + L) or goat anti-mouse IgG (H + L) (Cell Signaling Technology, USA). The results were visualized and analyzed by Odyssey Infrared Imaging System (LICOR, USA).