0.45 μm polyvinylidene difluoride membrane

Proteins were extracted using nuclear (Pierce) (Rockford, IL, USA) (for GATA-4) and total protein extraction reagents (Qiagen) according to the manufacturer’s protocol. The proteins were separated using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to 0.45 μm polyvinylidene difluoride membrane (Millipore, Burlington, MA, USA). The membranes were blocked with 5% skim milk in TBST at room temperature for 1 h and incubated with primary antibodies of anti-CD9, anti-calnexin (Abcam, Branford, CT, USA), anti-CD63 (Applied Biological Materials, Richmond, BC, Canada), anti-HSP70 (Cell Signaling, Danvers, MA, USA), anti-GATA-4 (Santa CruZ, Santa Cruz, CA, USA), and anti-THBS1 (Sigma-Aldrich, Saint Louis, MO, USA) at 4 °C overnight. After washing three times with TBST, the membranes were probed with HRP-conjugated secondary antibodies (Cell Signaling), respectively, at room temperature for 1 h and visualized using ECL Plus kit (GE Healthcare, Cincinnati, OH, USA). The expression of a specific protein was normalized with histone H3 and β-actin.

HEK293T cells were seeded at ~50% confluence the day before transfection in DMEM supplemented with 10% fetal bovine serum. Transfection was performed using Lipofectamine 2000. For each 10 cm dish, 200 μl of Plus reagent was diluted in 1 ml of OptiMEM (Life Technologies) with 12 μg of lenti-CRISPR plasmid library, 4 μg of pVSVG and 8 μg of psPAX2. Then, 30 μl of Lipofectamine 2000 was diluted in 1 ml of OptiMEM, and after 5 min, this solution was added to the DNA mixture. The complete mixture was incubated for 20 min before being added to cells. After 6 h, the medium was changed to 12 ml of DMEM. After 48 h, the medium was removed and centrifuged at 3 000 r.p.m. at 4 °C for 10 min to pellet the cell debris. The supernatant was filtered through a 0.45 μm polyvinylidene difluoride membrane (Millipore, Darmstadt, Germany). The MOI was measured as described in Shalem et al. [21 ].

The A549 cells were treated with various concentrations of resveratrol (25, 50 and 100 μm/l) for 48 h and lysed with radioimmunoprecipitation assay lysis buffer (Beyotime Institute of Biotechnology, Jiangsu, China). The cell lysates were centrifuged at 13,000 × gfor 10 min and the supernatant was collected. Protein concentration was determined using the Bradford protein assay (10 (link)). Total protein (20 μl) was separated on 10–15% sodium-dodecyl sulfate polyacrylamide gel electrophoresis and transferred onto a 0.45 μm polyvinylidene difluoride membrane (Millipore, Billerica, MA, USA) in a buffer containing 25 mmol/l Tris-HCl (pH 8.3), 192 mmol/l glycine and 20% methanol. The membranes were then blocked with 5% fat-free dry milk in Tris-buffered saline and Tween-20 containing 0.05% Tween-20 for 2 h and then incubated with primary antibodies overnight at 4°C. Subsequently, treatment with the appropriate horseradish peroxidase conjugated secondary antibody was performed at a dilution of 1:2,000. The immunoreactive bands were detected using the enhanced chemiluminescence method.

The human and mouse bone protein lysates were loaded into 10% SDS-PAGE gels, and the gels were cut into two parts. They were transferred into a 0.45-μm polyvinylidene difluoride membrane (Millipore) and separated. The large molecule protein CSPG4 (A3592, ABclonal) was processed at 300 mA for 3 h at 4°C with 10% methanol, and ITGA2B (A5680, ABclonal), tubulin (GB11017, Servicebio), and β-actin (GB11001, Servicebio) were processed at 300 mA for 1.5 h at 4°C with 20% methanol. The intensity of the protein was analyzed with ImageJ software.

Total protein (20-40 μg) from human gingival tissues (n=4) was separated by 10% SDS–PAGE and then transferred onto a 0.45-μm polyvinylidene difluoride membrane (Millipore) at 250 mA for 2 h on ice. The membrane was blocked with 5% skim milk dissolved in Tris-buffered saline containing 0.05% Tween 20 and probed with primary antibodies against CD38 (cat# sc-374650), CD79A (cat# sc-20064), ADPGK (cat# sc-100751) were purchased from Santa Cruz Biotechnology (Dallas, Texas, USA), OGN (cat# 12755-1-AP), HLA-DPA1 (cat# 16109-1-AP), PLCH1 (cat# 19143-1-AP), GAPDH (cat# 10494-1-AP) were purchased from Proteintech (Wuhan, Hubei, P.R.C), TMED5 (cat# SRP08852), GSTCD (cat# SRP11511) were purchased from Saier Biotechnology (Tianjin, P.R.C) and TBXAS1 (cat# ab157481) was purchased from Abcam (Cambridge, MA, USA) at the indicated dilutions overnight at 4°C. The membrane was then incubated with horseradish peroxidase (HRP)–conjugated secondary antibodies (anti-rabbit, cat# B900210 and anti-mouse, cat#SA00001-1) were purchased from Proteintech (Wuhan, Hubei, P.R.C) and detected by enhanced chemiluminescence reagents (Biosharp Life Sciences) using an image analyser. Levels of target proteins were normalized to GAPDH, which served as a reference control. The intensity of the protein bands was analyzed with ImageJ software, and the values are expressed as the mean ± standard deviation (SD).