P47phox

2×10⁷ cells were lysed using 350–400 µL lysis buffer (Pierce, Rockford, IL) supplemented with protease and phosphatase inhibitors (Sigma, St. Louis, MO), and lysates were cleared by centrifugation at 13,000 rpm for 30 min at 4°C. Equal amounts of total protein lysates (15 µg) were resolved by SDS-PAGE, transferred onto PVDF membranes and probed with antibodies. c-Cbl (C-15) antibody was from Santa Cruz Biotechnology (Santa Cruz, CA). pS621c-Raf antibody was from Pierce Thermo Scientific (Lafayette, CO). Lyn, Fgr, pY416-SFK, AhR, Vav1, Slp76, p47^phox, c-Raf, pS259c-Raf, pS289/296/301c-Raf, VDR, RARα, GAPDH, horseradish peroxidase anti-mouse and horseradish peroxidase anti-rabbit were from Cell Signaling (Danvers, MA, USA). Enhanced chemiluminescence ECL reagent (GE Healthcare, Pittsburg, PA) was used for detection.

BMDMs or lungs were harvested at designated time points and homogenized in PBS containing 0.1% Triton X-100 (phosphatase and protease inhibitor cocktail added). After centrifugation the supernatants were used for immunoblotting. Total protein content in the supernatant was measured using a BCA protein assay kit (Thermofisher, NY) to ensure that equal amounts of proteins were loaded onto 10% SDS-PAGE gels. Proteins were transferred to polyvinylidene fluoride membrane according to the protocol provided by Bio-Rad. Appropriate primary antibodies against mouse NLRP6 (Sigma, MO), phospho-MLKL (Abcam, MA), RIP3, RIP1, P47^phox, P67^phox, gp91^phox, phospho-p38 MAPK, phospho-JNK, phospho-Stat3, caspase-8, GAPDH (Cell Signaling, MA), caspase-1 (Adipogen), and gasdermin-D (Santa Cruz, CA) were added to the membrane and incubated overnight at 4⁰ C. Appropriate secondary antibodies were used, and the films were developed using ECL plus western blot detection system (ThermoFisher, NY). IL-1β, TNF-α, IFN-γ, IL-1α, and IL-6 were measured in BALF supernatants by ELISA according to the manufacturer’s protocol (eBioscience, CA).

For co-immunoprecipitation assay, cells were washed with PBS and harvested in a cell lysis buffer (30 mM Tris, pH 8.0, 10 mM NaCl,5 mM EDTA, 10 g/l polyoxyethylene-8-lauryl ether, 1 mM 0-phenanthroline,1 mM iodoacetamide, 10 mM NaF, 5 mM orthovanadate, and 10 mM sodium pyrophosphate). After being precleared using 20 μl of protein A/G PLUS-agarose (Santa Cruz Biotechnology Inc.), cell lysates were incubated with the first protein-specific antiserum (e.g., anti-p47phox) overnight at 4°C and then were incubated with 20 μl of protein A/G PLUS-agarose for additional 2 h with rotation. All immunoprecipitated samples were washed with lysis buffer three times and subjected to SDS-PAGE, followed by immunoblot with the second antibody (e.g., NOX2 or AIP1). The chemiluminescence was detected using ECL kit (Merck Millipore).
For immunoblot in artery and culture cells, protein was extracted from homogenized tissues or cells in lysis buffer, and boiled in SDS sample buffer for 10 min. Equal amounts of protein per sample were separated by SDS-PAGE and electrotransferred to polyvinylidene fluoride membranes (Bio-Rad Laboratories). Antibodies against AIP1 were described previously (Zhang et al., 2003 (link)). Primary antibodies used included AIP1 (Invitrogen), NOX1, NOX2, NOX4, NOX5 (Abcam), p47phox, p22phox (Cell Signaling Technology), FLAG, HA and GAPDH (Sigma).

CSF1, GM-CSF, IL-4, Q-VD-OPh and Z-VAD-FMK were obtained from R&D Systems (Minneapolis, MN, USA). Ac-YVAD-cmk from InvivoGen (San Diego, CA, USA), staurosporine and cleaved caspase-3 blocking peptide (#1050) from Cell Signaling (Danvers, MA, USA), IDN-6556 from MedChemtronica (Stockholm, Sweden), Tiron, TEMPO and Mito-TEMPO from Santa Cruz (Dallas, TX, USA). We used antibodies to calreticulin (#ab22683, Abcam, Cambridge, MA, USA), active caspase-3 (#9664, Cell Signaling), caspase-3 (#7148, Santa Cruz), caspase-3 (#9662, Cell Signaling), caspase-7 (#9492, Cell Signaling), caspase-7 (#sc-81654, Santa Cruz), caspase-8 (#9746, Cell Signaling), caspase-8 (#AF705, R&D Systems), DIABLO (#2409, ProSci, Poway, CA, USA), FADD (#2782, Cell Signaling), GST (#sc-138, Santa Cruz), γ-H2AX (#05-636, Merck Millipore, Billerica, MA, USA), HSC70 (#ADI-SPA-816, Enzo Life Sciences, Farmingdale, NY, USA), mtHSP70 (#MA3-028, Thermo Fisher Scientific, Waltham, MA, USA), LC3 (#M152-3, MBL International, Woburn, MA, USA), NOX2 (#sc-130543, Santa Cruz), p47^PHOX (#4312, Cell Signaling), TOM20 (#sc-11415, Santa Cruz).

Cell lysates were treated with bicine chaps buffer containing 1x protease cocktail inhibitor (Roche, USA) and prepared for western blot analysis as previously described [21 (link)]. Blots were immunoblotted with different antibodies including Atox1 (1:1000), GAPDH (1:500 dilution), p84 (1:500 dilution), Cyclin D1 (1:500 dilution) and p47 phox (1:500 dilution) (Cell Signaling Technology, MA, USA) overnight at 4°C. After washing three times in TBST buffer, the membranes were exposed to HRP-conjugated secondary antibodies for 1 hour at room temperature and imaged using enhanced chemiluminescence to detect immobilized antibodies. Densitometric analysis of immunoblots for respective proteins (Atox1, GAPDH, Nuclear matrix protein (p84), Cyclin D and p47 phox) was done by using image J software. All the experiments were repeated at least three times under same conditions.

Whole cell lysates of HL-60 cells were prepared using 200 μL of a Pierce M-PER lysis buffer solution (Pierce, Rockford, IL) containing phosphatase and protease inhibitors (Sigma, St. Louis, MO). Lysates were centrifuged at 16,000 RCF for 30 min. Protein lysates were resolved by gel electrophoresis and transferred onto Immobilon-P PVDF membrane (Millipore Corporation, Billerica MA). Equal amounts of protein lysates (25 μg) were resolved by SDS-PAGE gel electrophoresis. For immunoprecipitation, equal amounts of protein (250 μg) were prECLeared with 30 μL of A/G protein beads (Santa Cruz Biotechnology, Santa Cruz, CA). Lysates were then treated 1/100 volume of antibody and 50 μL of beads overnight at 4 °C prior to electrophoresis. Blots were probed with antibodies: AhR (H211), c-Cbl (C-15)- (Santa Cruz Biotechnology, Santa Cruz, CA); CD38 (BD Biosciences, San Jose, CA); phospho-p44/42 MAPK (ERK1/2) (Thr202/Tyr204) (D13.14.4E), p44/42 MAPK (ERK1/2) (137F5), pS221 MEK1/2, MEK1/2, Lyn, Fgr, Vav1, p47^phox, c-Raf, pS259c-Raf, GAPDH (Cell Signaling, Danvers, MA). Horseradish peroxidase anti-mouse and anti-rabbit antibodies (Cell Signaling, Danvers, MA) and ECL (GE Healthcare, Pittsburgh, PA) were used during detection. Blots were repeated at least three times.