Goat anti rabbit igg h l hrp preadsorbed secondary antibody

Subcellular fractions were prepared from groups of five 10–15-week-old and 30-50-week-old wt and cTnI-G203S hearts, an NE-PER^TM Nuclear and Cytoplasmic Extraction Kit (ThermoFisher Scientific, 78833), supplemented with cOmplete^™, Mini, EDTA-free Protease (Roche, 4693159001) and PhosStop^TM phosphatase (Roche, 4906837001) inhibitor tablets, according to manufacturer protocols. Blots were probed with the following primary antibodies: Total mTOR, mTOR (7C10) rabbit mAb (Cell Signaling Technology, 2983, 1:1000); Active mTOR, phospho-mTOR (Ser2448) (D9C2) XP® Rabbit mAb (Cell Signaling Technology, 5536, 1:1000); Total SRP6, S6 ribosomal protein (5G10) rabbit mAb (Cell Signaling Technology, 2217, 1:1000); Active SRP6, phospho-S6 ribosomal protein (Ser235/236) (2F9) rabbit mAb (Cell Signaling Technology, 4856, 1:1000). The following primary antibodies were used for loading controls: β-tubulin antibody (Cell Signaling Technology, 2146, 1:500); histone H2B (D2H6) rabbit mAb (Cell Signaling Technology, 12364, 1:1000). Blots were probed with goat anti-rabbit IgG H&L (HRP) preadsorbed secondary antibody (Abcam, ab97080, 1:10000). The same antibodies were used to confirm purity of cytoplasmic and nuclear fractions (Supplementary Fig. 5).

Total protein was isolated from lung tissue homogenates. The lysate was centrifuged at 11,000 g for 1 min at 4°C. Supernatants were collected and the protein content was quantitated using a BCA Protein Assay Kit (Beyotime Institute of Biotechnology, China). Equal amounts of total protein were separated using 12% SDS polyacrylamide gels, and then transferred on to PVDF membranes (Millipore, USA). After blocking with 5% milk in TBS containing 0.05% Tween-20 (TBST) for 1 h at 37°C, membranes were incubated with rabbit anti-actin monoclonal antibody (1:1000 dilution, ab179467, Abcam), rabbit anti-VEGF Receptor 1 monoclonal antibody (1:2000 dilution, ab32152, Abcam), or rabbit anti-PGF polyclonal antibody (1:1000 dilution, TA332424, OriGene Technologies, USA) at 37°C for 1 h. After washing with TBST three times, all membranes were incubated with goat anti-rabbit IgG H&L (HRP) preadsorbed secondary antibody (1:5000 dilution) (Abcam) at 37°C for 40 min. After washing with TBST three times, protein expression was visualized using Immobilon Western Chemilum HRP Substrate (Millipore). Actin served as an internal loading control. Relative protein expression was calculated using a method described previously (25 (link)).