Bicinchoninic acid kit

Total proteins from cells and tissues were extracted, and the concentrations were determined using a bicinchoninic acid kit (Qiagen GmbH, Hilden, Germany) according to the manufacturer’s protocol. The extracted proteins were run on sodium dodecyl sulfate polyacrylamide gel electrophoresis with the voltage increasing from 80 V to 120 V. Then the proteins were transferred onto polyvinylidene fluoride membranes using the semi-dry method at 80 mV for 30–45 min. The membranes were incubated with 5% bovine serum albumin at room temperature for 1 h, and then incubated with the primary antibodies against OPG (1:1000, ab2302), receptor activator of nuclear factor kappa B (RANK, 1:1000, ab32370), RANK ligand (RANKL, 1:5000, ab32064) and β-actin (1:5000, ab227387) (all purchased from Abcam) at 4 °C overnight. Afterwards, the membranes were washed with tris-buffered saline tween (TBST) (3 × 5 min), and incubated with the corresponding secondary antibody horseradish peroxidase-labeled immunoglobulin G (ab6747, 1:10000, Abcam) at room temperature for 1 h, After 3 times of TBST washes (5 min for each), the bands were visualized using chemiluminescence reagent on a Bio-Rad Gel Dol EZ imager (Bio-Rad Laboratories, California, USA). The target band was analyzed by calculating the gray value using ImageJ software (National Institutes of Health, Bethesda, Maryland, USA).

Menthol (Sigma), Icilin (sigma) and 4-(3-Chloro-2-pyridinyl)-N-[4-(1,1-dimethylethyl) phenyl]-1-piperazinecarboxamide (BCTC) (Sigma) were solubilized in dimethyl sulfoxide (DMSO) to prepare a stock solution. Work solutions were prepared in the bath solution (for electrophysiological recordings) or culture medium (differentiation assays). In all experiments, final concentration of DMSO was <0.1%. StemPro Adipogenesis Differentiation Kit and StemPro Osteogenesis Differentiation Kit were purchased to Gibco. LipidTox, Goat anti-rabbit IgG, Anti Rabbit Alexa Fluor 488 and Lipofectamine were purchase to Invitrogen. The anti-TRPM8 antibody was from Alomone labs (ACC-049). RNeasy Micro Kit, SuperScript III, Bicinchoninic acid kit and protease/phosphatase inhibitor cocktail were purchase from Qiagen, Life Technologies, Sigma and Cell signaling, respectively. 100 bp DNA ladder, Polyvinylidene difluoride membrane (PVDF), SuperSignal West Dura Extended Duration Substrate and RIPA buffer were from Thermo Scientific.

Proteins were extracted from the indicated cells using a Beyotime protein extraction kit, followed by the quantification of the protein levels in these samples using a bicinchoninic acid kit (Qiagen, CA, USA). A total of 30 μg of protein per sample was then separated via 10% SDS‐PAGE and transferred onto PVDF membranes (Millipore Corp., Bedford, MA, USA). These blots were then blocked with 5% non‐fat milk at 37°C for 2 h, after which they were probed overnight with antibodies from Cell Signaling Technology (Danvers, MA) specific for human LC3 (1:500, Cat. #12741), P62 (1:1000, Cat. #88588), Beclin1 (1:500, Cat. #3495), P65 (1:500, Cat. #3034), p‐P65 (Ser536) (1:500, Cat. #3033), IkBkB (1:1000, Cat. #8943), p‐IkBkB (Ser176/180) (1:500, Cat. #2694), and GAPDH (1:1000, Cat. #2118) at 4°C. Blots were then incubated with appropriate HRP‐conjugated secondary antibodies (Abcam, Cambridge, MA, USA) for 1 h at 37°C, after which protein levels were quantified via enhanced chemiluminescence (Pierce Biotechnology, Rockford, IL, USA) using Image Lab 2.0 (Bio‐Rad Laboratories, Hercules, CA, USA).