Sc 8041

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).