Sodium dodecyl sulfate polyacrylamide gel

MDBK cells infected or not infected with LSDV-WT were washed three times with cold PBS at specific time points and then harvested with lysis buffer containing 1% protease inhibitors (Beyotime). The protein concentration was determined with a BCA Protein Assay Kit (Beyotime). The cell extracts were resolved with sodium dodecyl sulfate polyacrylamide gel electrophoresis and transferred to polyvinylidene difluoride membranes (Beyotime). Total ORF142 protein was detected with a rabbit anti-ORF142 antibody (stored in our laboratory), with rabbit anti-β-actin (Proteintech, Wuhan, China) as the loading control.

A standard protein extraction kit (Beyotime, Nanjing, China) was used to extract the total protein in the tissue homogenate and OB-6 cells. Bicinchoninic acid (Nanjing JianCheng Bioengineering) method was employed to determine the protein concentration. Equal volumes of protein samples (30 μg) were loaded on a sodium dodecyl sulfate-polyacrylamide gel (Beyotime, Nanjing, China), and transferred to polyvinylidene fluoride membranes (IPVH00010, Millipore, Bedford, MA, United States). The membranes were probed with primary antibodies against LC3B (1:2,000, ab192890), Beclin-1 (1:1,000, ab210498), TLR4 (1:1,000, ab95562), p38MAPK (1:1,000, ab31828), ERK (1:1,000, ab53277), JNK (1:1,000, ab124956), NF-κB (1:1,000, ab16502), and Tubulin (1:5,000, ab7291) (all from Abcam Inc., Cambridge, MA, United States). Then the membranes were probed with secondary antibody goat anti-rabbit immunoglobulin G (IgG). The protein bands were visualized using Odyssey infrared imaging system (Li-Cor Bioscience, Lincoln, NE, United States) and quantified by Image Pro Plus 6.0 (Media Cybernetics, Silver Spring, United States).

The western blot assay method has been previously described.^{59 (link)} NP cells were lysed in RIPA buffer containing protease and phosphatase inhibitors (Beyotime). After centrifugation (4 °C, 12 000 r·min⁻¹, 10 min), the supernatant was collected, and the total protein concentration was determined using the BCA Protein Assay Kit (Beyotime). Each sample (30 mg total protein) was separated in sodium dodecyl sulfate-polyacrylamide gel (Beyotime) and transferred to nitrocellulose filter membranes (Millipore). After blocking with western blocking buffer (Beyotime) for 1 h at 37 °C, the membranes were incubated with primary anti-BMAL1 (1:1 000), aggrecan (1:1 000), MMP13 (1:1 500), pMLC (1:1 000), and β-actin antibodies (1:2 000) at 4 °C overnight. The membranes were then incubated with the appropriate HRP-conjugated secondary antibodies (1:2 000) for 1 h at 37 °C. Finally, the bands were detected using ECL-Plus Reagent (Millipore) and observed under an Amersham Imager 600 (General Electric).

MC3T3-E1 cells were incubated in 6-well plates at a density of 5 × 10⁵ cells/well. Cells cultured in a medium containing different concentrations (0–200 μM) of β-ecdysterone and noggin (0.5 mg/ml) were included in the experimental and control groups, respectively. After 7 days of induction culture, proteins were extracted with cell lysis buffer; the protein concentration was normalized before transfer. Proteins denatured in equal amounts from different samples were separated by electrophoresis on 10% sodium dodecyl sulfate-polyacrylamide gel (Beyotime) and then transferred to a polyvinylidene fluoride membrane. After the protein transfer membrane was enclosed in blocking buffer (Tris-buffered saline containing 0.1% Tween 20 and 5% fat-free milk) for 1 h, it was incubated with primary antibody at 4°C overnight. The goat anti-rabbit antibody (Boster) was then incubated at 37°C for 2 h. The ChemiDoc XRS + chemiluminescence detection system (BIO-RAD) was used for observation and the strip strength was analyzed using ImageJ software. The primary antibodies used were BMP-2 (1:1,000, Abcam), Smad1/5 (1:1,000, Abcam), P-Smad1/5 (1:1,000, Abcam), Runx2 (1:1,000, Abcam), and Osterix (1:1,000, Abcam). All experiments were repeated thrice.

Radioimmunoprecipitation assay lysis buffer was used for cell lysis and total protein extraction. The total protein content was quantified with a bicinchoninic acid protein assay kit (Thermo Fisher Scientific). The protein extracts were combined with protein loading buffer and boiled for 10 min. Then, 15 μg of each protein sample was separated on a 10% sodium dodecyl sulfate polyacrylamide gel (80 V for 40 min, 120 V for 80 min) (Beyotime) and transferred to a polyvinylidene difluoride membrane (210 mA constant current, 60 min) (Millipore). The membrane was blocked with 5% skimmed milk at room temperature for 2 h and then incubated with anti-CDK4 (1:1000, Cell Signaling Technology, USA, 12790s) or anti-GAPDH (1:500, Bioss, China, bs-0755R) primary antibodies overnight at 4° C. The membranes were subsequently incubated with goat anti-rabbit IgG secondary antibodies conjugated to horseradish peroxidase (1:5000, Bioss, bs-0295G-HRP) at room temperature for 2 h. Finally, the proteins were visualized with a Western Bright ECL Kit (Advansta, USA).

Brain tissues and SPN neurons were extracted using protein extraction kit containing protease inhibitor cocktail and DTT (#K269, Biovision, Milpitas, CA). Phosphatase inhibitors (Roche, Germany) was added into the extraction to prevent the degradation of phosphorylated proteins. Protein concentration was determined by a commercial Bradford kit (Beyotime, Jiangsu, China).
Equal amounts of protein (20 μg) were loaded and separated by 10% sodium dodecyl sulfate–polyacrylamide gel (Beyotime, Jiangsu, China), and then transferred to polyvinylidenedifluoride membrane (Millipore, Billerica, MA, USA). After blocking with 5% nonfat milk in TBST for 1 h at room temperature, the membrane was probed with primary antibodies overnight at 4 °C and then was incubated with secondary HRP-conjugated antibodies for 2 h at room temperature. After three more washes, blots were coated with ECL 2 (Thermo Pierce) and exposed to ChemiScope Mini (Qingxiang, Shanghai, China). Phosphorated proteins and total proteins were analyzed with Chemi Analysis software. Immunoblotting experiments were repeated at least twice to ensure reproducibility. The detailed information of the primary and secondary antibodies is shown in Supplementary Data 3.