Horseradish peroxidase hrp conjugated goat anti rabbit igg secondary antibody

PBMCs from five healthy controls and five SLE patients were lysed using protein lysis buffer (Beyotime Institute of Biotechnology, Beijing, China) supplemented with protease inhibitor cocktail (Pierce, Rockford, IL, USA) at 4°C for 20 min. Protein samples were separated using 10% sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis and then electrophoretically transferred to polyvinylidene difluoride membranes (Millipore,Billerica, MA, USA). Anti-Wnt 16 antibody (ab109437, abcam, Cambridge, UK) was diluted with primary antibody dilution buffer (Beyotime Institute of Biotechnology) to 1:1,000, and anti-GAPDH antibody (GOOD HERE, Hangzhou, China) was also diluted 1:1,000. The membranes were then washed with TBST buffer five times for 5 min each and incubated with horseradish peroxidase (HRP)-conjugated goat antirabbit IgG secondary antibody (1:5,000 dilution) (MULTI SCIENCES) for 1.5 h at 37°C. Bands were detected using enhanced chemiluminescence and visualized with a Gel Doc 2,000 (BioRad, Hercules, CA, USA).

Forty-eight hours after NovelmiRNA-25 mimic transfection, HEK293T cells and PBMCs from 5 SLE patients and 5 HCs were lysed using protein lysis buffer (Beyotime Institute of Biotechnology, Beijing, China) supplemented with protease inhibitor cocktail (Pierce, Rockford, IL, USA) at 4 °C for 20 min. Protein samples were separated using 10% sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis and then electrophoretically transferred to polyvinylidene difluoride membranes (Millipore, Billerica, MA, USA). Antibodies against AMPD2 (ab31537, MULTI SCIENCES, Hangzhou, China) were diluted with primary antibody dilution buffer (Beyotime Institute of Biotechnology) at a 1:400 dilution, while those against GAPDH (GOOD HERE, Hangzhou, China) were diluted 1:1000. The membranes were then washed with TBST buffer five times for 5 min each and incubated with horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG secondary antibody (1:5000 dilution) (MULTI SCIENCES) for 1.5 h at 37 °C. Bands were detected using enhanced chemiluminescence and visualized with a Gel Doc 2000 (BioRad, Hercules, CA, USA).

MDSCs-derived IPCs were lysed with a RIPA lysis buffer (Takara Bio, Japan) for 15 min at 4°C to extract total protein. Protein concentrations were measured using a BCA Protein Assay Kit (Thermo Fisher Scientific, CA, USA). Total proteins were separated by SDS-PAGE and then transferred onto polyvinylidene difluoride (PVDF) membranes. Membranes were incubated with blocking buffer (5% nonfat dry milk dissolved in 1× Tris buffered saline with 0.1% tween-20 (1× TBST)) at room temperature for 1 h, followed by the primary antibodies (STK4, YAP1, p-YAP1, and GAPDH (1:1,000, Abcam, UK) at 4°C overnight. Then, membranes were incubated with horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG secondary antibody (1:500, MultiSciences, China) for 1 h at room temperature. Protein bands were visualized using ECL reagent kit (Thermo Fisher Scientific, CA, USA). Protein images were captured using a ChemiDoc^™ imaging system (Bio-Rad, CA, USA). GAPDH was used as a reference control.