Anti serca2a

Western blots to analyse protein expression were conducted using the standard protocol as described previously 17, 18. Primary antibodies used were anti‐L‐type Ca²⁺ channel (Abcam, Cambridge, UK), anti‐ryanodine receptor (Abcam), anti‐SERCA2a (Santa Cruz Biotechnology, Dallas, TX, USA), anti‐NCX (Cell Signaling, Danvers, MA, USA), anti‐phospho‐PLB (Millipore, Bellerica, MA, USA), anti‐total‐PLB (Millipore), anti‐PMCA1 (Abcam), anti‐PMCA4 (Abcam), anti‐RAGE (Abcam), anti‐phospho‐p38 (Cell Signaling), anti‐total‐p38 (Cell Signaling), anti‐iNOS (Abcam), anti‐α‐tubulin (Calbiochem) and anti‐GAPDH (Santa Cruz). For visualization, we used HRP‐conjugated secondary antibodies, which were obtained from either Cell Signaling or Dako.

The protein expression was examined by western blot analysis as previously described16 (link)19 (link). Total protein was harvested from cultured cells or myocardium tissue. Briefly, samples were separated on 8–12% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS–PAGE), and then transferred to a nitrocellulose membrane by electroblotting. The membranes were blocked in 5% bovine serum albumin for 3 h, and then incubated with first antibody anti-SERCA2a, anti-NCX1, anti-SUMO1, anti-p-PLB, anti-PLB (Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-Akt and anti-phospho-Akt (Cell Signaling technology, CST, USA). After overnight incubated at 4 °C, the membranes were then washed and incubated with second antibodies for another 1 h at room temperature. Bands were visualized by the use of a super-western sensitivity chemiluminescence detection system (Tanon Imaging System, Tanon, China). Autoradiographs were quantitated by densitometry (Tanon Imaging System, Tanon, China). GAPDH or β-actin was the internal control for protein normalization.
The expressions of apoptosis protein including Bcl-2, Bax, caspase-3 and cleaved-caspase-3 were also detected by western blot analysis. Anti-Bcl-2, anti-Bax, anti-caspase-3 and anti-cleaved-caspase-3 were brought from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

Immunofluorescence analyses were conducted using the protocol as described elsewhere 17, 18. To detect NFκB subcellular localization, we used anti‐NFκB (p65) (Santa Cruz) and anti‐α‐actinin (Sigma) as primary antibodies and detected using fluorescent‐labelled secondary antibodies (Jackson Lab, West Grove, PA, USA). DAPI was used to stain the nuclei. To examine S‐nitrosylation of RyR and SERCA2a, we used anti‐SERCA2a (Santa Cruz Biotechnology), anti‐RYR (Abcam) and anti‐S‐nitrosocysteine (Abcam).