The lysates from frozen tissues were separated by SDS-PAGE and transferred onto nitrocellulose membranes. After blocking with 5% milk for 2 h, membranes were incubated with the following primary antibodies at 4°C overnight: anti-BLVRA (1:1000, Santa Cruz), eNOS (1:1000, Santa Cruz), phospho-eNOS (S1177) (1:1000, Santa Cruz), iNOS (1:1000, Abcam), phospho-iNOS (Y151) (1:1000, Abcam), nNOS (1:1000, Abcam), phospho-nNOS (S1417) (1:1000, Abcam), CD36 (1:500, Santa Cruz), mannose receptor (CD206, 1:500, Santa Cruz), TLR4 (1:1000, Abcam), Actin (1:3000, Abcam), 3-Nitrotyrosine (3-NT, 1:1000, Abcam), 4-Hydroxynonenal (HNE, 1:1000, Abcam), dinitrophenol (DNP, 1:1000, Abcam), IL-1β (1:1000, Abcam), IL-6 (1:2000, Abcam), TNF-α (1:1000, Abcam), Lamin B (1:1000, Abcam), TLR2 (1:1000, Abcam), TLR3 (1:1000, Abcam), TLR9 (1:1000, Abcam), followed by respective secondary antibodies (1:5000, Santa Cruz) for 2 h then visualized by ECL plus Kit (GE Healthcare and Life Science). Relative density of the protein immunoblot images was analyzed by ImageJ software.
For cytosol-nuclei translocation study, NE-PER Nuclear and Cytoplasmic Extraction Reagents (Thermo Scientific) was used to isolate cytosolic and nuclear protein lysates according to manufacturer’s instructions. The following steps of Western blot were performed as described previously27 (link).
For cytosol-nuclei translocation study, NE-PER Nuclear and Cytoplasmic Extraction Reagents (Thermo Scientific) was used to isolate cytosolic and nuclear protein lysates according to manufacturer’s instructions. The following steps of Western blot were performed as described previously27 (link).