Mapk family antibody sampler kit

Recombinant mouse soluble RANKL (Gene ID: 21943) was obtained from R&D Systems (Catalog number: 462-TEC-010, Oakville, ON, Canada). Dulbecco modified Eagles medium (DMEM), fetal bovine serum (FBS), penicillin-streptomycin (10,000 U/mL), and TRIzol Reagent were purchased from Thermo Fisher Scientific (Burlington, ON, Canada). Ovotransferrin (conalbumin from chicken egg white) with purity ≥98% was purchased from Sigma-Aldrich (Catalog number: C0755, St. Louis, MO, USA). The tartrate-resistant acid phosphatase (TRAP)-staining kit was obtained from Sigma-Aldrich (Catalog number: 387A-1KT, St. Louis, MO, USA). The annexin V-FITC Apoptosis Staining/Detection Kit was purchased from Abcam (Catalog number: ab14085, Toronto, ON, Canada). The NF-κB pathway sampler kit (Catalog number: 9936T), MAPK family antibody sampler kit (Catalog number: 9926T), and phosphor-MAPK family antibody sampler kit (Catalog number: 9910T) were purchased from Cell Signaling Technology (Whitby, ON, Canada). Recombinant anti-TRAF6 antibody (Catalog number: ab33915), anti-c-Fos antibody (Catalog number: ab190289), and anti-α tubulin antibody (Catalog number: ab7291) were purchased from Abcam (Cambridge, MA, USA). NFATc1 antibody (Catalog number: 7A6) and cathepsin K antibody (Catalog number: E-7) were purchased from Santa Cruz Biotechnology (Dallas, TX, USA).

Rat anti-mouse bcl-2 and Bim antibody were purchased from Santa Cruz Biotechnology, Santa Cruz, CA. Recombinant HMGB1 was purchased from R&D System, Minneapolis, MI. Rat anti-mouse p53, phosphorylated p53, bcl-2 and Bax antibody were purchased from Cell Signaling Technology, Beverly, MA. MAPK family antibody sampler kit and phospho-MAPK family antibody sampler kit were purchased from Cell Signaling Technology, Beverly, MA. Annexin V- fluorescein isothiocyante (FITC) was purchased from BD, San Diego, CA. The p38 MAPK inhibitor (SB203580) was purchased from Selleck Chemicals, Houston, TX. Enzyme-linked immunosorbent assay (ELISA) kits of IL-12, IL-2, IL-4, interferon (IFN)-γ, and TNF-α were purchased from Biosource, Worcester, MA. Nuclear extract and nuclear factor of activated T cell (NF-AT) assay kits were purchased from Active Motif, Carlsbad, CA.

Cells were lysed in RIPA lysis buffer (Beyotime, Shanghai, China). The concentration of each protein sample was measured using a BCA assay kit (Sigma-Aldrich, St. Louis, MO, USA). The samples were separated by SDS-PAGE electrophoresis and transferred onto a PVDF membrane (Millipore, Billerica, MA, USA). The membrane was blocked with 5% dehydrated milk for 2 hours and then incubated with the primary antibodies at 4°C overnight. The information of primary antibodies used was as follows: RUNX2 (#12556, Cell Signaling Technology), EVL(#12536, Cell Signaling Technology), mitogen-activated protein kinase (MAPK) Family Antibody Sampler Kit (#9926, Cell Signaling Technology), Phospho-MAPK Family Antibody Sampler Kit (#9910, Cell Signaling Technology), DMP1 (PA5-88069, Invitrogen), DSPP (sc-73632, Santa Cruz), OSX (PA5-115697, Invitrogen), OCN (ab93876, Abcam), and GAPDH (#2118, Cell Signaling Technology). Proteins were quantified using the enhanced chemiluminescence kit (Pierce, IL, USA).

Western blot was done as previously described [19 (link)–21 (link)]. Primary antibodies used in the study were as follows: anti-TRPA1 (1:1000, Alomone), anti-TRPA1 (1:1000, Novus Biologicals, CO, USA), anti-TRPA1 (1:1000, LSBio, WA, USA), MAPK Family Antibody Sampler Kit (1:1000, Cell Signaling Technology), Phospho-MAPK Family Antibody Sampler Kit (1:1000, Cell Signaling Technology), anti-MKP-1 (1:1000, Thermo Fisher Scientific), PGC-1α (1:1000, Abcam), anti-β-actin (1:1000, Abcam), anti-β-tubulin (1:1000, Cell Signaling Technology). Secondary antibodies used were: HRP-conjugated goat anti-rabbit secondary antibody (1:3000, Dako), HRP-conjugated goat anti-mouse secondary antibody (1:3000, Dako).

Cells were harvested and lysed in RIPA buffer (Cell Signaling Technology, Danvers, MA, USA) containing protease inhibitor cocktail (Sigma-Aldrich). Proteins samples were separated using 10–15% SDS-PAGE and then transferred to membranes (Millipore, Burlington, MA, USA), which were then probed with primary and secondary antibodies. Membranes were developed using a chemiluminescence solution (GE Healthcare, Chicago, IL, USA) in a LAS-4000 Lumino-imaging unit (Fujifilm, Tokyo, Japan). Immunoblot band intensities were quantified using NIH ImageJ software (Version 1.53t, Fujifilm), and results were presented as intensity ratios versus β-actin. The antibodies used were as follows; anti-β-actin (Santa Cruz Biotechnology, Dallas, TX, USA), anti-human iNOS (Santa Cruz Biotechnology), anti-human TLR2 (Santa Cruz Biotechnology), anti-human TLR4 (Santa Cruz Biotechnology), NF-κB (Cell Signaling Technology), MAPK Family antibody sampler kit (Cell Signaling Technology), anti-human AIM2 (Cell Signaling Technology), anti-human ASC (Cell Signaling Technology), pro-Caspase-1 (Cell Signaling Technology), anti-human IL-1β (R&D Systems, Minneapolis, MN, USA), Beclin1 (Santa Cruz Biotechnology), ATG5 (Santa Cruz Biotechnology), and LC3 (Cell Signaling Technology).

After the indicated treatments, the proteins from the RA FLS were extracted using a cell lysis buffer. The protein concentration was determined with the Bradford assay (Bio-Rad Protein Assay; Bio-Rad, CA, USA). Protein samples (10–20 µg) were resolved in 8% gradient SDS-PAGE, transferred onto PVDF membranes (Merck Millipore, Darmstadt, Germany) and probed with primary antibodies directed against WNT5A (R&D), p44/42, SAPK/JNK, and p38 MAPK (MAPK Family antibody sampler kit, #9926), AKT (#9272), GSK3β(#9315), phospho-p44/42, phospho-SAPK/JNK, and phospho-p38 MAPK (Phospho-MAPK Family antibody sampler kit, #9910), phospho AKT (#9271), phospho GSK-3β (#9322), all from Cell Signaling Technology, Danvers, USA; and GAPDH antibody (Sigma-Aldrich). Bound antibodies were revealed with horseradish peroxidase-conjugated secondary antibodies (Cell Signaling and Santa Cruz Biotechnology), and blots were developed using the ECL Plus detection system (ChemiDoc™ MD Imaging System (Bio-Rad, California, EEUU)).