Sc 8436

Cells were placed in quiescing media (containing 1% FBS) until reaching confluency. VSMCs were treated with peptides (50 µM) for 24 h for the detection of AT₁R and MasR; ECs were treated with peptides (50 µM) for 18 h for the detection of TNFα receptors 1/2 (TNF-R1/R2). Peptide concentrations and time of treatment were selected per our previous studies [28 (link),33 (link)]. After the treatment, cells were scraped and lysed in a boiling Laemmle’s buffer with 50 mM DTT and 0.2% Triton-X-100.
Cell samples were run in a 9% separating gel and transferred to a nitrocellulose membrane before being incubated with specific primary antibodies. Protein bands of AT₁R (PA5-20812, Invitrogen), MasR (NBP1-78444, Novus Biologicals, Toronto, ON, Canada), TNF-R1 (sc-8436, Santa Cruz, Dallas, TX, USA), TNF-R2 (sc-8041, Santa Cruz), glutathione peroxidase 4 (GPx4; ab125066, Abcam, Toronto, ON, Canada), and superoxide dismutase 2 (SOD2; ab227091, Abcam) were normalized to α-tubulin (ab15246, Abcam) or glyceraldehyde 3-phosphate dehydrogenase (GAPDH, ab8245, Abcam). The fluorescent bands were visualized by adding donkey-anti-mouse IRDye 680 RD or donkey-anti-rabbit 800 CW secondary antibodies (Licor Biosciences, Lincoln, NE, USA), and the signals were detected using Licor Odyssey BioImager (Licor Biosciences).

Cell protein homogenates (25 μg) were separated by electrophoresis in 12% polyacrylamide gels, transferred to nitrocellulose membranes, and blocked overnight with 5% nonfat dry milk. The membranes were washed and incubated in the presence of the following monoclonal antibodies: anti-IL-1R1, anti-IL-1R2, anti-TNFR1, anti-TNFR2, anti-VDR (sc-393998, sc-376247, sc-8436, sc-393614, and sc-13133, respectively; Santa Cruz Biotechnology, CA, USA), and anti-glyceraldehyde 3-phosphate dehydrogenase (GAPDH, MAB374, Millipore, Milford, MA, USA) overnight at 4°C. Membranes were incubated in the presence of secondary antibody conjugated with horseradish peroxidase (sc-2031, Santa Cruz Biotechnology) for 2 hours at room temperature. The immunoblots were visualized by chemiluminescence using ECL Plus (Amersham Pharmacia, UK).