Sds polyacrylamide gel electrophoresis

According to the manufacturer's instructions, total cell lysates were extracted with RIPA lysis buffer containing 0.1% PMSF (Beyotime Biotech, China) and denatured at 100°C for 10 min. Total protein was separated by SDS-polyacrylamide gel electrophoresis (Beyotime Biotech, China) and then transferred onto PVDF membranes at 100 V for 80 min. Subsequently, the membranes were blocked with TBST containing 5% nonfat dry milk at room temperature for 60 min and incubated with the corresponding antibody overnight at 4°C followed by a horseradish peroxidase-conjugated secondary antibody (1 : 10000) for 60 min. Immunoreactive proteins were identified by Clarity™ Western ECL Substrate (Berkeley, California, USA). Densitometric analysis of the band intensity was performed using Image Lab software (Bio-Rad).

The following reagents were used in the study: Dulbecco’s modified Eagle’s medium (DMEM) and fetal bovine serum (FBS) were obtained from Wisent (Montreal, QC, Canada). Puromycin (PM), penicillin, and streptomycin were purchased from Sigma (St. Louis, MO, USA). SDS-polyacrylamide gel electrophoresis and 10% buffered neutral formaldehyde were purchased from Beyotime (Shanghai, China). Nitrocellulose membranes were provided by Millipore (Billerica, MA, USA). FITC-labeled anti-mouse F4/80, PE-conjugated anti-mouse programmed death ligand 1 (PD-L1), and PE-Cy5-labeled anti-mouse CD80 monoclonal antibodies were obtained from eBioscience (San Diego CA, USA) for flow cytometry analysis. Antibodies against MMP-2, MMP-9, and GAPDH used for Western blotting were manufactured by Proteintech (Chicago, IL, USA). Rat anti-mouse F4/80, CD68, IL-10, TGF-β, VEGF, and anti-mouse major histocompatibility complex class II (MHCII) monoclonal antibodies that recognize cells from both BALB/c and C57 BL/6 mice were manufactured by Abcam (Cambridge, MA, USA) for immunohistochemistry assays. The TNF-α enzyme-linked immunosorbent assay (ELISA) kit was provided by CUSABIO (Wuhan, China). The interleukin-6 (IL-6) ELISA kit was obtained from RayBiotech (Norcross GA, USA). The Greiss Reagent System determined for nitrite was purchased from Promega Biotech Company (Madison, WI, USA).

Cells or tissues were lysed with RIPA buffer (Beyotime, Hangzhou, China) containing a mixture of protease inhibitors. The total protein was quantified using BCA protein analysis kit (Beyotime). SDS-polyacrylamide gel electrophoresis (Beyotime) was used to separate the protein cleavage products. The main monovalent antibodies used were as follows: Cbl-b (1:500, Proteintech, Chicago, IL, USA), Atrogin-1 (1:1000, Bioss Antibodies, Beijing, China), MuRE-1 (1:1000, Proteintech). Protein was detected with BeyoECL Star reagent (Beyotime).

Raw 264.7 macrophages were seeded into a 6 cm plate (1.5 × 10⁶/well) and cultured overnight. LPS (0.2 μg/mL) and a series of concentrations of FLs were cotreated with cells for 24 h. Then, cells were lysed with radio-immunoprecipitation assay (RIPA) lysis buffer containing 1% of protease and phosphatase inhibitors (Beyotime, Shanghai, China). The total protein concentrations were determined using a bicinchoninic acid (BCA) protein assay kit (Beyotime, Shanghai, China), and equal amounts of protein were loaded on 10% of SDS-polyacrylamide gel electrophoresis (Beyotime, Shanghai, China). Subsequently, proteins were transferred to PVDF membranes (Merck Millipore, Darmstadt, Germany) and then blocked in the skim milk for 1 h at atmospheric temperature. The primary antibodies against β-actin, iNos, Cox-2, IκBα, p-P65, p-P38, p-ERK, and TLR4 (Cell Signaling, Boston, MA, USA) were incubated in the membrane overnight at 4 °C, respectively. The immunoreactive bands are visualized on a gel imaging system (Tanon Science & Technology, Shanghai, China) after incubation of the HRP-secondary antibody.

The grouping of transfection treatments was described above. Forty-eight hours after transfection, the total proteins were extracted from CP-II cells, and their concentrations were then determined using a bicinchoninic acid (BCA) protein assay reagent kit (Transgen, Shanghai, China). Equal amounts of protein were separated by 12% SDS-polyacrylamide gel electrophoresis (Beyotime, China) and blocked with 5% skim milk for 1 h. Then, primary antibodies for rabbit anti-MKP1 (ABclonal, A2919), p-ERK (ABclonal, AP0472), p-JNK (ABclonal, A4867), P38 (ABclonal, A0227), GAPDH (Abmart, M20024) or β-actin (Abmart, T40104) (at 1:5000 dilution) protein were incubated overnight at 4 ℃. Finally, the membrane was incubated with secondary antibody for 1 h after Tris-buffered saline with Tween-20 (TBST) (a 1× concentrated solution of Tris-buffered saline with Tween-20 with a concentration of 10 mM Tris-HCl, 15 mM NaCl, 0.05% Tween-20 at pH 7.5) washing. The enhanced chemiluminescence (ECL) detection system (Bio-Rad) was used to detect protein expression.

Forty-eight hours after transfection, DF-1 cells were lysed in RIPA buffer (Beyotime, Nantong, China) supplemented with 100 mM phenyl methane sulfonyl fluoride (PMSF) to exact total protein. Protein concentrations were measured by the Pierce BCA Protein Assay Kit (Transgen, Shanghai, China). An equal amount of protein was separated by 12% SDS-polyacrylamide gel electrophoresis (Beyotime, China) and blocked with 5% skim milk for 1h. Then, the membranes were separately probed with p-JUN (ABclonal, AP0048), p-FOS (ABclonal, AP0038), p-JNK1 (ABclonal, AP0631), Bcl-2 (ABclonal, A19693), Caspase8 (ABclonal, A0215), Caspase9 (ABclonal, A18676), Caspase3(ABclonal, A19654), GAPDH (Abmart, M20024) overnight at 4°C with a final dilution of 1:5000 (v/v). Finally, the membrane was incubated with the secondary antibody for 1h after TBST washing. The enhanced chemiluminescence (ECL) detection system (Bio-Rad) was used to detect protein expression.