Lrrc8c

Cells were stimulated with TNFα (10ng/mL). Protein extracts (40 μg) were separated by electrophoresis on a polyacrylamide gel (10%) and transferred to nitrocellulose membranes. Nonspecific binding was blocked with Odyssey blocking buffer (LI-COR Biosciences, Lincoln, NE) for 1 hour at room-temperature. Membranes were incubated with primary antibodies overnight at 4°C. Antibodies included: Tubulin (Vanderbilt Antibody Core), LRRC8A (#A304–175A, Bethyl Laboratories, Montgomery, TX), LRRC8C (HPA029347, Sigma-Aldrich), LRRC8D (HPA014745, Sigma-Aldrich), PCNA (05–347, Millipore). Signals were developed using fluorescent secondary antibodies with the Odyssey Imaging System (LI-COR Biosciences, Lincoln, NE) and quantified densitometrically.

Human aoritic VSMCs were stimulated with TNFα (10 ng/mL, 10 min) then lysed (50mM Tris base, 150mM NaCl, 1mM EDTA, 10% glycerol, 1mM DTT, 1% Triton X-100, 0.1% Na-DOC, 0.1% SDS, 10mM β-glycerophosphate, 20mM para-nitrophenyl phosphate, 2mM sodium pyrophosphate, 1mM Na₃VO₄, 5mM NaF, 10 μg/ml aprotinin, and 1 mM phenylmethylsulfonyl fluoride (PMSF) at pH 7.4) for 1 hour with nutation at 4°C, and centrifuged for 30min at 20,000g. Supernatants were pre-cleared with protein-G sepharose beads for 1h at 4°C and cleared-supernatants were incubated with antibody (2 μg) for 1.5h, then incubated with protein-G sepharose for 1h. Beads were washed with lysis buffer, resuspended in SDS sample buffer, boiled and the associated proteins were then analyzed by western blot. Antibodies for IP were used as follows; Nox1 (sc-5821, Santa Cruz), LRRC8C (HPA029347, Sigma-Aldrich), LRRC8D (HPA014745, Sigma-Aldrich).