Nfatc1

Phil (C₂₇H₃₄O₁₁, Purity ≥ 98%) was obtained from Chengdu Mansite Pharmaceutical Co. (Chengdu, Sichuan, China) (Figure 1F). Recombinant mouse M-CSF and RANKL were purchased from R&D Systems (Minneapolis, MN, USA). Dexamethasone, β-glycerophosphate, ascorbic acid, MTS reagents, tartrate-resistant acid phosphatase (TRAP) staining kit, and LPS were all gained from Sigma-Aldrich (St. Louis, MO, USA). Alkaline phosphatase (ALP) staining kit was obtained from Beyotime Biotechnology Inc. (Shanghai, China). TRIzol reagent was purchased from Invitrogen (Invitrogen Life Technologies, Carlsbad, CA, USA). Primary antibodies targeting phosphorylated extracellular signal-regulated kinase (p-ERK), phosphorylated c-Jun N-terminal kinases (p-JNK), phosphorylated p38 (p-p38), total ERK, total JNK, total p38, inhibitor of NF-κB (IκBα), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were from CST (Cell Signaling Technology, Inc., Beverly, MA, USA). Antibodies including c-Fos and NFATc1 were purchased from BD Biosciences (San Jose, CA, USA).

The cells were lysed in lysis buffer (50 mM Tris. Cl, 150 mM NaCl, 1% NP-40, 0.5% Na.deoxycholate, 0.1% SDS, and a protease inhibitor cocktail, phosphatase inhibitor cocktail). The equal amounts of proteins were separated by SDS-PAGE and transferred membrane (Whatman Protran, Dassel, Germany). The membrane was blocked and then incubated with primary antibodies (1:1000) such as NFATc1 (BD Pharmingen San Diego, CA, USA), c-Fos and Actin (Santa Cruz Biotechnology, Santa Cruz, CA, USA) followed by secondary antibodies (1:10000) (Jackson ImmunoResearch, West Grove, PA, USA). The proteins attached to the membrane was measured using enhanced chemiluminescence (ECL) detection system (Santa Cruz Biotechnology, Santa Cruz, CA, USA), according to the manufacturer’s instructions.

Total cell extracts were obtained using 1X RIPA lysis buffer (Cell Signaling, MA, USA) containing 1X proteinase-inhibitor cocktail (Calbiochem, CA, USA), 1 mM PMSF (Sigma-Aldrich), and 1X phosphatase-inhibitor (Cell Signaling, MA, USA). Cell lysates were fractionated on 10% SDS-polyacrylamide gels, transferred to nitrocellulose membranes (GE Healthcare Life Sciences), and incubated with specific primary antibodies. Immunoblotting was performed with the following antibodies: B7–H3 (R&D System); c-Fos (Abcam, MA, USA); NFATc1 (BD Biosciences, CA, USA); c-Fms, IκBα, p-p38 MAPK, p-p44/42 MAPK, p44/42 MAPK, IDO, p-STAT1, STAT1, AhR, β-actin, and GAPDH (Cell Signaling, MA, USA); RANK, p38α, and iNOS (Santa Cruz, CA, USA). Secondary antibodies (Jackson ImmunoResearch) were incubated before adding the ECL substrate (Invitrogen). For quantitative analysis of protein expression, the optical densities of the blot bands were measured using the Image J software.

A rabbit polyclonal P-Panx3 antibody was raised against a synthetic peptide (amino acid residues, SCFpSPSNFSC) from the first extracellular loop of the mouse Panx3 protein and the native peptide was used as a control sequence (SCFSPSNFSC). Rabbit anti-Panx3 antibody was used as previously described^{5 (link),16 (link)}. The Panx3 expression vector (pEF1/Panx3) and the control vector (pEF1) have been described previously^{16 (link)}. In brief, the pEF1/Panx3 vector was constructed by cloning the coding sequence of mouse Panx3 cDNA into the pEF1/V5-His vector (Invitrogen). The antibodies were obtained for Ocn from Biomedical Technology; CaMKII and P-CaMKII from Cell Signaling Technology; P-NFATc1 from Santa Cruz Biotechnology, Inc.; V5 from Invitrogen; and NFATc1 from BD. HA was from COVANCE; α-tubulin from Sigma-Aldrich, CIP from New England Biolabs; Apyrase from Sigma-Aldrich; LY294002 from Invitrogen; BMP2 from Humanzyme; and iQ SYBR Green Supermix from Bio-Rad Laboratories. ER-tracker was obtained from Invitrogen. HRP-conjugated goat anti–mouse and goat anti–rabbit IgG were obtained from United States Biological. The Akt-CA was obtained from Addgene. The inhibitory Panx3 peptide was described previously^{16 (link)}.

The extracts from cultured cells and total tissue were prepared using RIPA buffer or a lysis buffer [150 mmol·L⁻¹ Tris-HCl (pH 6.8), 6% SDS, 30% glycerol, and 0.03% bromophenol blue] with 10% 2-ME. The lysates were subjected to 7.5% SDS-PAGE and transferred to Immobilon-P membranes (Millipore, Billerica, MA, USA). The transferred membranes were blocked with 5% skimmed milk and incubated with specific antibodies. The following primary antibodies were used: SLIT3 (1:1 000; R&D), Nfatc1 (1:1 000; BD Pharmagen), c-Fos (1:1 000; Santa Cruz), p38 (1:3 000; Santa Cruz), and HSP90 (1:2 000; Sigma). We detected protein bands using Western Lightning plus-ECL (PerkinElmer, Waltham, MA, USA).