Horseradish peroxidase conjugated goat anti rabbit igg secondary antibody

M1/M2 polarization‐related protein expression was analyzed using Western blot. Briefly, macrophages were isolated from the peritoneal cavity of rats as previously described.17 The isolated macrophages were homogenized as previously described.18 The protein concentration was determined by a Bradford assay (Bio‐Rad Laboratories, Hercules, CA, USA). Ten microgram aliquots of cell homogenate were loaded per well on a 10% sodiumdodecyl sulfate polyacrylamide gel electropheresis (SDS‐PAGE) gel. After transferring the protein to the membranes, they were then blocked with 5% non‐fat dried milk for 1 h and incubated with anti‐CD38 (1:1000 dilution), Egr2 (1:1000 dilution), IRF4 (1:1000 dilution), IRF5 (1:1000 dilution) and β‐actin antibody (1:1500 dilution) (Cell Signaling, Danvers, MA, USA) overnight at 4 °C followed by incubation with horseradish peroxidase‐conjugated goat anti‐rabbit IgG secondary antibody (Cell Signaling) for 2 h at room temperature. An electro‐chemi‐luminescence (ECL) chemiluminescence substrate (Amersham, Little Chalfont, UK) was used to visualize the immune reaction. The images were taken using ChemiDoc XRS+ system (Bio‐Rad Laboratories, Hercules, CA, USA) and analyzed using Image Lab 4.1 (Bio‐Rad Laboratories, Hercules, CA, USA).

Cellular proteins were extracted using cold RIPA lysis buffer (Beyotime, Shanghai, China) containing a protease inhibitor cocktail. Proteins were separated on 10% sodium dodecyl sulfate polyacrylamide gels and then transferred onto polyvinylidene difluoride membranes. The membranes were blocked with 5% nonfat dried milk and then incubated with primary antibodies (anti–NECTIN-1, 1:1000; Abcam) overnight at 4°C, followed by incubation with horseradish peroxidase–conjugated goat anti-rabbit IgG secondary antibody (1:1000; Cell Signaling Technology, Danvers, USA). Chemiluminescence solution (MilliporeSigma, Burlington, USA) was used to reveal the bands. Representative blots from at least three independent experiments were shown.

After treating with 10% FBS, Second-generation chondrocytes cultured with DMEM supplemented with 0.05% FBS were treated with 10 ng/ml IL-1β, or co-incubated with 10 ng/ml IL-1β and 1 mM AG for 2 h at 37˚C. Subsequently, the cells were washed with PBS and fixed with 4% paraformaldehyde for 1 h at 37˚C. The cells were washed with PBS, blocked with 10% goat serum (cat. no. AR1009; 0.3% Triton, 1:10; Boster Biological Technology) for 1 h at room temperature and rinsed with PBS. Subsequently, the cells were incubated with primary antibodies against p-p65 (cat. no. 3033; 1:100; Cell Signaling Technology, Inc.) overnight at 4˚C. Following primary incubation, the cells were gently washed and then incubated with a horseradish peroxidase-conjugated secondary antibody goat anti-rabbit IgG (cat. no. ZB-2301; 1:250; OriGene Technologies, Inc.) for 2 h at 37˚C. Subsequently, the cells were stained with DAPI (Beyotime Institute of Biotechnology) at room temperature for 15 min. Cells were rinsed and observed under a fluorescence microscope in six randomly-selected fields (magnification, x400).

U87 cells were lysed in pre-cooled RIPA lysis buffer (Beyotime Institute of Biotechnology). Protein quantification was performed using bicinchoninic acid protein concentration determination assay (Thermo Fisher Scientific, Inc.). Following incubation in a water bath for 10 min, 25 µg protein/lane were subjected to SDS-PAGE on 12% gels. The proteins were transferred to a PVDF membrane (EMD Millipore), blocked in 5% skim milk at 25˚C for 2 h and incubated with primary antibodies (all 1:1,000) against PRDX3 (cat. no. ab73349; Abcam), autophagy-related protein 9 (ATG9; cat. no. ab108338; Abcam), Ras-related protein Rab-1 (RAB1; cat. no. 13075; Cell Signaling Technology, Inc.), p62 (cat. no. ab91526; Abcam) and GAPDH (cat. no. 5174; Cell Signaling Technology, Inc.) overnight at 4˚C. The membranes were subsequently washed 3 times with PBS containing 5% Tween-20 and incubated with goat anti-rabbit IgG horseradish peroxidase-conjugated secondary antibody (1:2,000; cat. no. ab6721; Abcam) at 25˚C for 1 h. The membranes were treated with ECL reagent (EMD Millipore) to detect the expression of proteins using an automatic chemiluminescence analyzer (Tanon 5200; Tanon Science and Technology Co., Ltd.). The band gray values were read using the Tanon GIS 4.2 software (Tanon Science and Technology Co., Ltd.).

Total tissue and cell proteins were obtained using radioimmunoprecipitation assay buffer lysis buffer with proteinase inhibitors (protease inhibitor cocktail; Sigma-Aldrich, St. Louis, MO, USA). Equivalent amounts of proteins (30 µg) were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred onto nitrocellulose membranes (Invitrogen; Thermo Fisher Scientific, Inc.). Subsequent to blocking with 5% skimmed milk for 30 min, the membranes were incubated with a rabbit polyclonal anti-SNF2H primary antibody (dilution, 1:500; catalog no., ab3749; Abcam, Cambridge, MA, USA) at 4°C overnight. A goat anti-rabbit IgG horseradish peroxidase-conjugated secondary antibody (dilution, 1:3,000; catalog no., 7074; Cell Signaling Technology, Inc., Danvers, MA, USA) was used for the subsequent incubation at room temperature for 1 h. In addition, a mouse monoclonal β-actin horseradish peroxidase-conjugated antibody (dilution, 1:5,000; catalog no., ab20272; Abcam) was used as a loading control. The expression of SNF2H was detected on X-ray film using the ECL detection system (Pierce; Thermo Fisher Scientific, Inc.).