Lrrc8a

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.

Cells were serum-deprived (0.5% serum) for 3 h and then stimulated with TNFα (10 ng/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 h at room-temperature. Membranes were incubated with primary antibodies overnight at 4 °C. Antibodies included: p-JNK (#9255, Cell Signaling Technology, Danvers, MA), JNK (#9252, Cell Signaling Technology), p-ERK (#9101, Cell Signaling Technology), ERK (#9102, Cell Signaling Technology), Tubulin (Vanderbilt Antibody Core), iNOS (#610328, BD Transduction, San Jose, CA), VCAM (#AF643, R & D Systems, Minneapolis, MN), LRRC8A (#A304-175A, Bethyl Laboratories, Montgomery, TX), p22phox (#sc-271968, Santa Cruz Biotechnology, Dallas, TX), Nox1 (#SAB2501686, Sigma-Aldrich). Signals were developed using fluorescent secondary antibodies with the Odyssey Imaging System (LI-COR Biosciences, Lincoln, NE) and quantified densitometrically.

Cells were stimulated with TNFα (10 ng/mL) then lysed (0.01 M Tris base, 1 mM EDTA, 30 mM NaCl, 1% Nonidet P40, protease inhibitor cocktail, and phenylmethylsulfonyl fluoride (PMSF) at pH 7.4) for 1 h with nutation at 4 °C, and centrifuged for 30 min at 20,000g. Supernatants were pre-cleared with protein-G sepharose beads for 1 h at 4 °C and cleared-supernatants were incubated with antibody (2 μg) for 1.5 h, then incubated with protein-G sepharose for 1 h. 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: LRRC8A (#A304-175A, #A304-173A, Bethyl Laboratories), p22phox (#sc-20781, #sc-11712, Santa Cruz Biotechnology).