Radioimmunoprecipitation assay (ripa)

Total protein extraction was implemented using radio-immunoprecipitation assay (R0010, Solarbio) containing phenylmethylsulfonyl fluoride. The protein concentration was detected by bicinchoninic acid kit (C503021-0500, Shanghai Sangon Biological Engineering Technology & Services Co., Ltd., Shanghai, China). Next, 50 μg of protein was separated with sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE), and electrotransferred onto a polyvinylidene fluoride membrane (Merck Millipore, USA). Membrane blockade was conducted using 5% BSA on a shaking table for 1 h, and then the membrane was incubated overnight at 4 °C with the following primary antibodies: rabbit anti-PER2 (ab179813, 1:5000), Runx2 (ab23981, 1:1000), OCN (ab93876, 1:500), β-catenin (ab32572, 1:5000), C-Myc (ab32072, 1:1000), Cyclin D1 (ab16663, 1:4000), Wnt7b (ab94915, 1:1000), GAPDH (ab8245, 1:5000, internal reference), and histone H3 (ab1791, 1:1000). All antibodies were provided by Abcam. Subsequently, the membrane was incubated with the horseradish peroxidase-labeled goat anti-rabbit secondary antibody immunoglobulin G (IgG) (ab6721, 1:5000, Abcam) for 1 h. Following three TBST washes (15 min per wash), the membrane was added with the luminescent solution. The results were analyzed by Bio-Rad gel imaging analysis system (Bio-Rad, Hercules, CA, USA) and Image J.

Extraction of total protein was made from the lung cells using RIPA (Solarbio, Beijing, China) buffer and centrifuged at 12,000× g. Cell protein was separated by 10% SDS-PAGE, and then transferred onto polyvinylidene fluoride (PVDF) membranes. The membranes were blocked with 10% skimmed milk (P0216, Beyotime, Shanghai, China) and incubated with primary antibodies. The monoclonal antibody was obtained from Abcam (Cambridge, MA, USA). HRP conjugated AffiniPure goat anti-rabbit IgG (H + L) was obtained from Proteintech (Proteintech Group, Rosemont, IL, USA). Detailed antibody information is in the supplementary file.

Total protein was extracted by RIPA (Solarbio, Beijing), and the concentration was measured by bicinchoninic acid (Solarbio). The nuclear and cytoplasmic fractions were isolated by a Nuclear Extraction Kit (Solarbio, Beijing, China) according to the manufacturer‘s instruction. 80 μg/well proteins were loaded onto 10% SDS-PAGE for each sample, and proteins were transferred onto equilibrated polyvinylidene difluoride membranes (Millipore Corporation, Billerica, MA, USA) by electroblotting. Membranes were blocked with TBST containing 5% milk and incubated overnight at 4°C with primary antibodies against HO-1 (Bioworld Tech, LP, USA), Wnt1 (Abcam, Cambridge, MA, USA), Wnt5a (Novus Biologicals, Littleton, USA), β-catenin, GSK-3β, phospho-GSK-3β (Ser9) (ProteinTech Group, Chicago, USA), and NFAT5 (Santa Cruz, CA, USA). After incubation with the secondary antibody (ProteinTech), proteins expression was corrected by the amount of β-actin (ProteinTech) in the same sample. Lamin A (ProteinTech) and tubulin (ProteinTech) were used as the marker for nuclear and cytosolic proteins, respectively. The bands were quantified by scanning densitometry using Quantity One 4.6.3 software (Bio Rad).

Total protein was isolated from LX‐2 cells using RIPA (Solarbio) and quantified using a BCA Protein Assay Kit (Solarbio). Then equal amounts of protein samples were separated on SDS‐PAGE and transferred to polyvinylidene difluoride membranes. The membranes were blocked with 5% skimmed milk for 60 minutes, and incubated with primary antibodies against CAPRIN1 (1:500; Proteintech), Cyclin D1 (1:1000; Bioss, Beijing, China), Cyclin E1 (1:1000; Proteintech), p‐RbS807 (1:1000; Bioss), TGF‐β2 (1:1000; Bioss), COL1A1 (1:1000; Bioss), α‐SMA (1:1000; Bioss), p‐Smad2^ser467 (1:1000; Cell Signaling Technology, Trask Lane Danvers, MA), Smad2 (1:1000; Cell Signaling Technology), p‐Smad3^S423/S425 (1:1000; Cell Signaling Technology), Smad3(1:1000; Cell Signaling Technology) and β‐actin (1:1000; Santacruz Biotechnology) at 4°C overnight. Goat anti‐rabbit IgG or goat anti‐mouse secondary antibodies (1:3000; Solarbio) were employed at 37°C for 60 minutes. The bands were visualized using electro‐chemi‐luminescence (ECL) Western Blotting Substrate (Solarbio). The protein expression was standardized to β‐actin and quantified using Gel‐Pro‐Analyzer software (Media Cybernetics, Rockville, MD).

BMMs or RAW264.7 cells were seeded (4 × 10⁵ cells/well) in 6-well plates. Cells were pretreated with or without 200-nM SB2390663 for 3 h. Cells were then treated with 50 ng/ml RANKL for indicated time, with or without 200-nM SB239063 for 30 min or 48 h. All the proteins were obtained from cultured cells by using RIPA (Solar bio, Beijing, China) supplemented with 100-mM phenylmethanesulfonyl fluoride (Beyotime, Zhengzhou, China), 100×Phosphatase Inhibitor Cocktail (CWBIO, China), and Protease Inhibitor Cocktail (Millipore, USA). After the 15-min centrifugation at 12,000 rpm, supernatants were extracted. Proteins were resolved on 10% SDS-PAGE and transferred by electroblotting to PVDF membranes (Millipore, USA). Membranes were then blocked in 5% (w/v) nonfat dry milk in TBS with Tween 20 (TBST) at RT for 45 min, followed by incubation with indicated antibodies (1:1,000 dilution) at 4°C overnight. Washed five times with TBST, bands were then incubated with the HRP-conjugated goat anti-mouse/rabbit IgG (1:5,000 dilution; Abcam). Bands were detected by Image Lab software (Bio-Rad, Hercules, CA). The images were quantified by Image J.