Horseradish peroxidase linked anti rabbit igg secondary antibody

Samples were subjected to Western blot analysis as previously described (Qadhi et al., 2013 (link)). Briefly, H9c2 cell samples (20 µg protein) were resolved in 12% SDS-polyacrylamide gel and then transferred electrophoretically to nitrocellulose membranes, that were blocked in TBS-T buffer (0.15 M NaCl, 3 mM KCl, 25 mM trishydroxymethyl methylamine and 0.1% tween-25, pH 7.4) with 5% skim milk for 2 h at room temperature. Membranes were washed four times with TBS-T buffer, and then incubated with a phosphor-Akt antibody for Ser473 (1:500, Cell Signaling, cat #9271) overnight at 4 °C. Membranes were washed as described above and then incubated with horseradish-peroxidase linked anti-rabbit IgG secondary antibody (1:10,000, Cell Signaling, cat #7074) for 2 h at room temperature. A similar procedure was used to probe membranes for total Akt (1:1000, Cell Signaling, cat #9272S), and we utilized GAPDH (1:1000, Cell Signaling, cat #2118S) and ULK-1 (1:1000, Cell Signaling, cat #8054) as loading controls. Chemiluminscence solution was used to detect signals. Relative band intensity was expressed in arbitrary units, and was assessed by densitometry using Image J software (NIH, Bethesda, MD, USA).