α tubulin

Mouse periodontal soft tissues and BMDMs were homogenized manually and lysed in radio immunoprecipitation assay (RIPA) buffer (Beyotime Biotechnology, China) containing protease inhibitor and phosphatase inhibitor. All samples were quantified and normalized by bicinchoninic acid (BCA) kit (Thermo Fisher Scientific). Protein extracts were loaded into sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene fluoride (PVDF, Millipore, USA) membranes, which were then blocked in 5% skim milk for 1 h at room temperature (RT). The membranes were incubated with primary antibodies against Tim4 (1:1 000, Sigma-Aldrich), Igf1 (1:1 000, ABclonal, China), p-p38 (1:1 000, CST, USA), p38 MAPK (1:1 000, CST), and α-Tubulin (1: 5 000, ABclonal) overnight at 4 °C, followed by anti-rabbit horseradish peroxidase (HRP)-conjugated secondary antibody (1: 10 000, Proteintech, China, 1:5 000) for 1 h at RT. The results were visualized with a chemiluminescence imaging system and further analyzed by ImageJ software. Relative protein expression levels normalized to α-Tubulin were evaluated.

BODIPY 493/503 (#D3922) and DAPI (#D1306) were purchased from Invitrogen (CA, USA). SKL2001 was purchased from Selleckchem (Shanghai, China). Dexamethasone (#D4902), 3-isobutyl-1-methylxanthine (#I5879), insulin (#I2643), Oil red O (#O9755), MS-222 (#E10521), and Triton X-100 (#T8787) were purchased from Sigma-Aldrich (MO, USA). Antibodies against P42/44 (#9102), β-catenin (#9562), Phospho-β-catenin (#9561), PPAR-γ (#2443), Ubiquitin (#3936), GAPDH (#5174), HA-tag (#3724) as well as secondary HRP-conjugated antibodies against mouse (#7076) and rabbit (#7074) were purchased from CST (MA, USA) and used at 1:1,000 dilution for western blotting. Anti-FLAG-tag antibody (#F1804) and anti-FLAG-tag M2 affinity gel (#A2220) were from Sigma-Aldrich (MO, USA). Antibodies against Plin1 (#A16294), OSBPL2 (#A14199), α-Tubulin (#AC012), β-catenin (#A11512) as well as secondary HRP-conjugated Mouse Anti-Rabbit IgG Light Chain (#AS061) were from ABclonal (Wuhan, China). Antibodies against β-catenin (#BF0319) for zebrafish were from Affinity Biosciences (Changzhou, China). Alexa Fluor 488 donkey anti-mouse IgG and Alexa Fluor 546 donkey anti-rabbit IgG were purchased from Life Technologies (CA, USA).

Cells were homogenized in RIPA butter supplemented with protease and phosphatase inhibitors using a cell scraper. Lysates were centrifuged at 15,000 × g for 15 min at 4 °C. Protein concentration was quantified using a bicinchoninic acid (BCA) assay. A total of 30 µg from each sample were loaded into each lane and separated by electrophoresis on a 10% SDS polyacrylamide gel. After electrophoresis, proteins were transferred to cellulose nitrate membranes. Nonspecific binding was blocked through incubation with PBST (PBS with 0.1% Tween 20) containing 5% skim milk for at least 1 h at room temperature. Membranes were incubated with antibodies including TGR5 (1:1000, Abcam, catalog No: ab72608) and α-Tubulin (1:3000, ABclonal, catalog No: AC012). For detection, HRP-linked anti-rabbit and anti-mouse IgG secondary antibodies were used and a chemiluminescent signal was detected with a digital imager (Biotanon, Tanon 5200).

The Nuclear and Cytoplasmic Protein Extraction Kit (Beyotime, Shanghai, China) was used for nuclear and cytoplasmic proteins extraction. Cell were harvested and lysed in radio immunoprecipitation assay (RIPA) buffer (Beyotime, Shanghai, China) with protease inhibitor phenylmethylsulfonyl fluoride (PMSF) (Beyotime, Shanghai, China). The lysates were centrifuged at 10,000 g, 4 °C, for 15 min. Protein concentration was determined by Ultra-micro spectrophotometer (Implen, Munich, Germany). Proteins were separated on 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto polyvinylidene difluoride (PVDF) membranes. Membranes were blocked with 5% non-fat milk for 1 h and incubated with primary antibodies overnight at 4 °C. Then the membranes were washed with Tris-Buffered Saline Tween20 (TBS-T) and incubated with secondary antibody for 1.5 h. Chemiluminescence imaging system (Tanon 5500, Shanghai, China) was used to detect the membranes. Protein levels were standardized by comparison with Lamin B (Santa Cruz, Dallas, CA, USA) or α-tubulin (ABclonal, Boston, MA, USA). Representative blots were chosen from three independent experiments.