P mypt

Western blot analysis was performed as previously described37 (link). For these experiments, we used a mouse monoclonal antibody against nNOS (1:2000, BD Biosciences), a rabbit polyclonal antibody against nNOS phosphorylated In Ser1417 (P-nNOS, 1:2000, Abcam), a rabbit monoclonal antibody against Rho kinase 1 (ROCK1, 1:1000, Abcam), a mouse monoclonal antibody against ROCK2 (1:500, Abcam), a rabbit polyclonal antibody against myosin phosphatase (MYPT, 1:2000, Abcam), a rabbit polyclonal antibody against MYPT phosphorylated In Thr696 (P-MYPT, 1:500, Abcam), and a monoclonal anti-β-actin-peroxidase antibody (1:50000, Sigma-Aldrich, Spain). Appropriate positive controls were used for each blot.

The concentration of total protein of each sample was determined twice with a BCA protein assay kit (Applygen Technologies), according to the manufacture’s instruction, taking the average as the concentration. The protein was mixed with 2x electrophoresis sample buffer, separated on 8% or 10% SDS-PAGE, and transferred to polyvinylidene difluoride membrane. The membrane was blocked with 3% skimmed milk powder, rinsing with TBS-Tween for three times, 5 min each, and then cut and incubated overnight at 4 °C with antibodies, respectively, against glyceraldehyde-3-phosphatedehydrogenase (GAPDH), p-MLC, MLC, Bcl-2, Bax, cleaved-Caspase-3, cleaved-Caspase-9, cTnI (1:1000, Cell Signaling Technology, Berley, MA, USA), RhoA, ROCK1, MYPT, p-MYPT (1:1000, Abcam, Cambrige, MA, USA), ATP5D (1:200, Santa Cruz, California, USA). Following rinsing three times, 5 min each, the membrane was incubated with secondary antibody for 1 hour at room temperature, rinsed with TBS-Tween three times, 10 min each. The Quantification of target protein was carried out by scanning densitometry in the X-film using a bio-image analysis system (Image-Pro plus 6.0; Media Cybernetic, Bethesda, MD, USA)13 (link).