Anti p mypt1 t696

Hearts from the euthanized mice were fixed in 4% formaldehyde (pH 7.3) for 12 h, dehydrated in alcohol, clarified in xylene, and embedded in paraffin to be sectioned at 5 μm. Protein levels were evaluated in cardiac tissues using the immunoperoxidase technique with anti-p-MYPT1 (T696) (Cell Signaling, USA #5163), anti-ROCK1 (Cell Signaling, USA #4035), anti-ROCK2 (Cell Signaling, USA #9029) antibodies. Staining was performed using a peroxidase and diaminobenzidine kit with a chromophore according to the manufacturer’s instructions (RTU-Vectastain kit; Vector Laboratories, USA). The heart tissue was additionally stained with hematoxylin. The images were obtained with a Nikon Eclipse 400 epifluorescence microscope (Nikon, Japan) and analyzed with ImageJ software (ImageJ 1.47v).

U937 cells were washed with PBS and lysed as described previously (27 (link)). Total protein concentration was quantified with Bradford Assay. Extracted proteins were separated by SDS-PAGE (100 V), and transferred to a nitrocellulose membrane (110 V, 90 min). Membranes were blocked in 5% (w/v) non-fat milk powder in TBS-T for 1 h at room temperature (RT), washed with TBS-T, and incubated with primary antibody overnight at 4°C. Membranes were washed and incubated with HRP-conjugated secondary antibodies for 2 h at RT, then washed and incubated with enhanced chemiluminescence (ECL) reagents (Sigma-Aldrich, USA) according to the manufacturer’s instructions. The primary antibodies used were anti-ROCK1 (Cell Signaling, USA #4035), anti-ROCK2 (Cell Signaling, USA #9029), anti-MYPT1 (Cell Signaling, USA #2634), anti-pMYPT1 (T696) (Cell Signaling, USA #5163), anti-p65 (Cell Signaling, USA #8242), anti-p-p65(Ser536) (Cell Signaling, USA #3033), anti-GAPDH (Cell Signaling, USA #2118), and anti-β-actin (Cell Signaling, USA #8457). Secondary antibodies used were anti-mouse-HRP (Santa Cruz Biotechnology, USA SC-516102) and anti-rabbit-HRP (Santa Cruz Biotechnology, USA SC-2357).