Horseradish peroxidase hrp conjugated goat anti rabbit or anti mouse igg

Protein from the sixteen samples (four groups with n = 4 per group) used in RT-qPCR was extracted from spleens with Cell Lysis Buffer for Western & IP (Beyotime, Shanghai, China) and used for expression analysis by Western blotting. Protein concentration was measured with a BCA kit (Beyotime). A 75 μg aliquot of each protein sample was loaded and then separated in SDS-PAGE 10% gels (Fdbio science, Hangzhou, China), and transferred to polyvinylidene fluoride membranes (Solarbio, Beijing, China). After blocking for 2 h at room temperature in Western Blocking Buffer (Beyotime), the membranes were incubated overnight at 4 °C with rabbit polyclonal antibody against CXCL13 (1:100 dilution; GenScript, Nanjing, China) or mouse anti-GAPDH monoclonal antibody (1:1000 dilution; Beyotime). The membranes were washed with PBS containing 0.05% Tween-20 (PBST) and incubated with horseradish peroxidase (HRP)-conjugated goat anti-rabbit or anti-mouse IgG (1:1000 dilution; Beyotime) at room temperature for 2 h. The resulting signals were visualized using FDbio-Dura ECL solution A and FDbio-Femto ECL solution B (Fdbio Science), collected by Fusion FX system (Vilber Lourmat, Marne-la-Vallée, France), and measured with ImageJ software [24 ].

For performing Western blot analysis, the cells were washed with PBS and lysis buffer (P0013B, Beyotime). After incubation on ice for 30 min, the homogenate was centrifuged at 12,000g for 15 min at 4 °C, and the supernatant was used for protein analysis. Protein concentration was determined using the BCA protein assay (P0012S, Beyotime, Beijing, China). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis was performed using the Bio-Rad Mini-Protean Tetra System (Bio-Rad, Hercules, CA, US). The proteins on the gel were then transferred onto polyvinylidene fluoride membranes (PVDF, Millipore, Beijing, China) for 2 h at 200 mA and blocked for 2 h with 5% skim milk at room temperature to prevent nonspecific binding. The blots were probed with the indicated primary antibodies followed by horseradish peroxidase (HRP)-conjugated goat anti-rabbit or anti-mouse IgG (Beyotime). Rabbit anti-Bcl-2 and mouse anti-actin antibodies were purchased from Beyotime. Rabbit anti-Bax, anti-Caspass-3, and anti-PARP monoclonal antibodies were purchased from Cell Signaling Technology (Danvers, MA, USA) [28 (link)].