Chemiluminescent detection system

RAW264.7 cells were seeded into 6-well plates at a density of 5 × 10⁵ per well, and treated with different concentrations of YPF for 24 h. Then the cells were lysed with RIPA lysis buffer, and quantitated using BCA protein reagent assay kit (YEASEN, China) and analyzed by 8% of SDS-PAGE, followed by immunoblotting using enhanced chemiluminescence substrate (Merck Millipore) according to the manufacturer’s instructions. Bands were visualized using a chemiluminescent detection system (Bio-Rad). The expression of β-actin protein was used as a standard.

Proteins were extracted from gastric tissues or GES-1 cells using radioimmunoprecipitation assay (RIPA) lysis buffer (Beyotime, Shanghai, China), and protein quantification was conducted using the BCA method (Beyotime, China). Equal amounts of proteins were then separated on a 10% SDS-PAGE gel and subsequently transferred to a polyvinylidene difluoride (PVDF) membrane. Following blocking for 10 min at room temperature using rapid blocking solution (Ncmbio, Suzhou, China), PVDF membranes were incubated with primary antibodies overnight at 4 °C. Afterward, the PVDF membrane was washed three times using tris−buffered saline (TBST) and incubated with secondary antibody for 1 h at room temperature. Finally, emitter-coupled logic (ECL) substrate (Ncmbio, China) was added dropwise, and the membrane was observed using a chemiluminescent detection system (Bio-Rad, Hercules, CA, USA). Quantification of protein bands was performed using Image J software (1.53K). The antibodies used were as follows: β-actin (AB0035, 1:5000, Abways, Beijing, China), Bax (A20227, 1:2000, ABclonal, Wuhan, China), Bcl2 (A20777, 1:1000, ABclonal, China), TNF−α (ER1919-22, 1:500, HuaBio, Hangzhou, China), IL−6 (R1412-2, 1:500, HuaBio, China), and NF−κB (PSH0−27, 1:1000, HuaBio, China).

NFs and KFs were divided into three groups Control (“C”, only treated with medium), Vehicle (“V”, medium 0.55% DMSO), and TA (550 μM TA dissolved in 0.55% DMSO with medium) for 48 h exposure separately. Proteins were extracted from each cell line and the concentrations were measured by BCA assay. Sixty micrograms of each sample were mixed with 10 μL of 5X protein sample loading buffer. The trans-blotted PVDF membrane was then blocked with blocking buffer (3% BSA; 1X TBS; and 0.05% Tween-20) for 1 h. Afterward, It was incubated with rabbit monoclonal anti-human collagen type I (dilution 1 : 2000, Thermo Fisher Scientific, USA), over two nights at 4°C. Anti-rabbit HRP-conjugated secondary antibody (dilution at 1 : 20000, Sigma, USA) was then added to the membrane on a shaker for an hour at room temperature. After rinsing with TBST, the membrane was activated with chemiluminescent HRP substrate (Cell Signaling Technology, USA) for 4 min in the dark at room temperature. It was visualized and quantified using a chemiluminescent detection system (Bio-Rad, USA). Protein band intensity in each lane was scored by volume intensity and was normalized to beta-actin (dilution at 1 : 4000, Sigma, USA) [27 (link)]. All experiments were performed independently. Four times with keloid fibroblast cultures culture as well as normal skin dermal culture.

Total protein extracts were produced using RIPA buffer including PMSF protease inhibitors (Beyotime Biotechnology, China) and a phosphatase inhibitor (Beyotime Biotechnology, China) from homogenized tissues or cultured cells. An Enhanced BCA Protein Assay Kit was used to measure protein (Beyotime Biotechnology, China). SDS-PAGE was used to separate equal quantities of protein before wet transfer onto 0.45 mm/0.22 mm polyvinylidene difluoride (Merck Millipore, Billerica, MA). The membranes were blocked for 2 h at room temperature with 5% non-fat milk. After blocking, the membranes were incubated with primary antibodies against IGF2BP3 (1:1000; ABclonal, China, Cat: #A6099), PRL (1:1,000; ABclonal, China, Cat: #A1618), IGFBP1 (1:1,000; cst, Cat: #31025S), TGFb1 (1:1,000; ABclonal, China, Cat: #A15103), or GAPDH (ABclonal, China, Cat: #AC036) overnight at 4°C. Then, the secondary antibodies were used (1:5,000; ABclonal, China, Cat: #ASO14) to incubate the membranes for 1 h at room temperature.
The protein expression was detected by the chemiluminescent detection system (Bio-Rad, United States) once adding the ECL Enhanced Plus Kits (RM0021P, ABclonal, China). Protein expression was analyzed using Image Lab Analyzer software.