Nfatc1

BMMs were seeded into six-well plates at the appropriate density and stimulated with 30 ng/mL M-CSF and 50 ng/mL RANKL plus either MSNs (16 or 64 μg/mL) or MSNs-ISL (16 or 64 μg/mL) for 30 min or 24 h. The cells were washed twice with ice-cold PBS and then lysed using RIPA lysate containing protease and phosphatase inhibitor. Protein content was determined using a BCA protein quantification kit (Beyotime Inc, Shanghai, China) following the manufacturer's protocol. Total proteins were transferred onto a polyvinylidene difluoride membrane, blocked in non-fat milk for 1 h at room temperature, and incubated overnight with the specific primary antibodies at 4 °C. GAPDH served as the protein internal standard and the standard for quantifying protein expression. The specific primary antibodies used were p38, p-p38, extracellular signal-regulated protein kinase (ERK), p-ERK, nuclear factor-κB (NF-κB) p65, p NF-κB p65, inhibitor of κBα (IκBα), p-IκBα, GAPDH (Cell Signaling Technology, Danvers, MA), c-Jun N-terminal kinase (JNK), p-JNK, tumor necrosis factor receptor-associated factor 6 (TRAF6), and nuclear factor of activated T cells (NFATc1; Santa Cruz Biotechnology, Santa Cruz, CA). The gray values of the band were quantified.

Human macrophage colony-stimulating factor (M-CSF) and RANKL was purchased from Peprotech (Rocky Hill, NJ, United States). Rapamycin was obtained from Calbiochem. Antibodies were used for immunoblotting as follows: NFATc1 (Santa Cruz Biotechnology; sc-7294); α-tubulin (Sigma-Aldrich; T9026); phosphorylated p70S6K, p70S6K (Cell Signaling Technology; 9234, 2708); and GADD34 (Proteintech; 10449-1-AP). A mouse CTX-I and P1NP ELISA kits were purchased from Cloud-Clone (Wuhan, China).

Cells were lysed with RIPA Lysis buffer (Pierce Biotechnology, Rockford, IL, USA). The protein concentration in the supernatants was determined using the Bradford method [24 (link)]. Protein samples (30 μg) were separated in sodium dodecyl sulfate-polyacrylamide gels and transferred to polyvinylidene fluoride membranes (GE, Buckinghamshire, UK) using a western blot apparatus. Each membrane was blocked in blocking buffer (2% bovine serum albumin or 5% skim milk) and then incubated with primary antibody ( phospholipase C gamma 2 (PLCγ2), p-ERK, p-JNK, p-p38, cAMP response element binding (CREB), p-IκBα, p-PLCγ2, ERK, JNK, p38, CREB (Cell signaling Technology, Danvers, MA, USA), NFATc1, c-fos (Santa Cruz Biotechnology, Santa Cruz, CA, USA), β-actin (Sigma-Aldrich)). Horseradish peroxidase-conjugated IgG (1:2000 dilutions) was used as the secondary antibody. Immunoreactivity was detected using a Mini HD6 image analyzer (Uvitec Cambridge, UK).