Anti traf6

Mouse lung tissue homogenates were separated by electrophoresis on 10 to 15% SDS-PAGE, transferred on PVDF membranes (Merck Millipore, Burlington, MA, USA), blocked in 5% of non-fat dry milk, and incubated with primary antibodies including anti-CitH3 (1:1000, citrulline R2 + R8 + R17, Abcam, Cambridge, UK), anti-TRAF6 (1:1000, Santa Cruz), anti-IRAK-1 (1:1000, Santa Cruz), anti-PAD4 (1:1000, Abcam), anti-HMGB1 (1:1000, Abcam) or anti-β-actin antibodies (1:5000, Sigma-Aldrich) at 4 °C for 18 h. After washing, the membranes were incubated with HRP-conjugated secondary antibodies (1:10,000, Jackson Immunoresearch, West Grove, PA, USA) at RT for 2 h. Signal expression of protein-antibody complexes was detected by ECL system (Amersham Pharmacia Biotech, Buckinghamshire, UK) and visualized with Biospectrum imaging system (UVP, Upland, CA, USA). Relative protein expression levels were measured by Image J (National Institute of Health, Bethesda, MD, USA).

Peroxidase-conjugated anti-rabbit and anti-mouse antibodies were purchased from Millipore (Billerica, MA, USA). Dulbecco’s modified Eagle’s medium (DMEM), fetal bovine serum (FBS), fetal calf serum, and lipofectamine and PLUS reagents were purchased from Life Technologies (Grand Island, NY, USA). Anti-Myc, anti-GAPDH, anti-TAK1, anti-TRAF6 and anti-IRAK1 antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Polyclonal anti-RCAN1 antibodies were purchased from ECM Biosciences (Versailles, KY, USA) and Santa Cruz Biotechnology. Anti-LRRK2, anti-phospho-LRRK2 (Ser935), and anti-phospho-TAK1 (Thr187) antibodies were purchased from Abcam (Cambridge, MA, USA) and Cell Signaling Technology (Beverly, MA, USA), respectively. Polyclonal and monoclonal anti-HA antibodies were purchased from Abnova (Tebu, France) and Covance (Powhatan, VA, USA), respectively. Protein A-Sepharose beads and glutathione sepharose 4B were purchased from GE Healthcare Life Science (Piscataway, NJ, USA). Enhanced chemiluminescence (ECL) reagent and [γ-32P] ATP were purchased from Perkin Elmer Life Sciences (Downers Grove, IL, USA). MG132 was purchased from A.G. Scientific (San Diego, CA, USA). Human recombinant IL-1β was purchased from Sigma (St. Louis, MO, USA).

Cells were harvested in lysis solution containing 50 mM Tris/HCl (pH 7.6), 1 % NP40, 150 mM NaCl, 2 mM EDTA, 100 μM PMSF, a protease inhibitor cocktail (Roche Applied Science, Basel, Switzerland), and a phosphatase inhibitor (Sigma-Aldrich). After incubation on ice for 30 min, cellular debris was removed by centrifugation (10 min, 4 °C). Proteins (10 μg) were separated by SDS-PAGE and then transferred to a polyvinylidene difluoride membrane. The following antibodies were used: anti-β-actin (Sigma-Aldrich), anti-TG2 (Neomarkers, Fremont, CA), anti-E-cadherin (Santa Cruz Biotechnology, Santa Cruz, CA), N-Cadherin (Santa Cruz Biotechnology), anti-phospho-NF-kB p65 (S276) (Cell Signaling, Danvers, MA), anti- NF-kB p65 (Cell Signaling), anti-I-kB (Santa Cruz Biotechnology), anti-phospho-JNK (Santa Cruz Biotechnology), anti-JNK (Santa Cruz Biotechnology), anti-IRAK1 (Cell Signaling), anti-IRAK2 (Cell Signaling), and anti-TRAF6 (Santa Cruz Biotechnology).

Total proteins were obtained by using radio immunoprecipitation assay (RIPA) buffer (Thermo Scientific, Waltham, MA, USA) containing a protease inhibitor cocktail (protease inhibitor cocktail 1 tablet; Roche Diagnostics, Mannheim, Germany) and centrifuged at 13,000 rpm for 10 min. The protein was measured with BCA Protein Assay Kit by using microplate reader at 562 nm. Equal amounts of protein were separated on 10% SDS-PAGE gel electrophoresis and transferred to nitrocellulose membranes (Bio-Rad Laboratories, Hercules, CA, USA). After blocking with 5% bovine serum albumin, protein was probed with appropriate antibodies.
The primary antibodies used were as follows: anti-TRAP, anti-cathepsin K, anti-c-Fos, anti-NFATc1, anti-TRAF6, and anti-β-actin from Santa Cruz Biotechnology (Santa Cruz, CA, USA); anti-carbonic anhydrase II, anti-IκBα, and anti-phospho-IκBα from Abcam (Cambridge, MA, USA); and anti-IL-1β and anti-cleaved IL-1β from Cell Signaling Technology (Danvers, MA, USA). The SuperSignal® West Pico chemiluminescent kit (Thermo Scientific, Rockford, IL, USA) was used to detect the target protein. Images were obtained using a ChemiDoc TM XRS system (Bio-Rad Laboratories, Hercules, CA, USA).

Sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) was used to separate the lysates (30 μg/lane) of lung and cell culture protein, which were then transferred to a polyvinylidene fluoride membrane (Millipore). The blots were incubated overnight with primary antibodies, including anti-Axl, anti- phosphorylated-Axl (1:1000, Bioss Inc., Beijing, China), anti-TNF receptor associated factor 6 (anti-TRAF6) (1:200, Santa Cruz Biotechnology, USA), anti-SOCS3, anti-phosphorylated-NF-κB p65, anti-IκB-α, anti-TATA (1:1000, Cell Signaling Technology, USA), and anti-β-actin (1:10000, Sigma Chemical Company, USA).

The plasmid of pcDNA4-myc/his-RNF183 was constructed by the company of genewiz (Suzhou, China). A truncated form of RNF183 without amino acids 1–59 was generated by PCR and subsequent molecular cloning into pcDNA4-myc/his vector. IL-8 and IL-8-△NF-κB reported plasmids were kindly provided by Professor Hongbin Shu (Wuhan University, China). The antibodies used were listed as follows: human anti-RNF183 antibody (1:1000 for western blot, 1:200 for IHC, ab197321, Abcam, Cambridge, UK), anti-TAB1 antibody (1;1000, #3226 CST), anti-TRAF2 antibody (1:200, sc-136999, Santa Cruz Biotech, Santa Cruz, CA, USA), anti-TRAF6 (1:200, sc-7221, Santa Cruz), anti-NF-κB p65 antibody (1:1000 for western blot, 1:200 for IHC, 1:100 for CHIP, #8242, CST), anti-NF-κB P50 antibody (1:1000, #12540, CST, Boston, MA, USA), anti-ki-67 antibody(1:1000, #12202, CST), anti-β-actin (1:200, sc-47778, Santa Cruz), anti-mouse IgG HRP conjugate (1:8000, W402B, Promega, Madison, WI, USA), anti-Rabbit IgG HRP conjugate (1:2000, #7074P2, CST), normal rabbit IgG (1 μg for ChIP, sc-3888, Santa Cruz). Recombinant human IL-8 was purchased from Novus (Littleton, CO, USA) (NBP2-34905). The siRNAs (ribobio Company, Beijing, China) sequences were listed in supplementary table 1.