Ripa protein extraction kit

The hippocampal tissues were isolated from the mice brain after animal behavior experiment. A RIPA protein extraction kit (Beyotime Biotechnology, China) was used for total protein extraction. The Pierce Mem-PER Eukaryotic Membrane Protein Extraction Kit (Pierce, USA) was used to extract membrane protein. Samples boiled with 4× sample buffer at 95°C for 5 min, and then separated on 8–10% sodium dodecyl sulphate-polyacrylamide gel electrophoresis gels and transferred onto polyvinylidene difluoride membranes (Millipore, USA). The target proteins were immunoblotted with primary antibodies overnight at 4°C and then incubated with HRP-conjugated secondary antibodies. The following primary antibodies were used: protrudin (rabbit, Proteintech, USA), GADPH (rabbit, Proteintech, USA), GABA_ARβ2/3 (mouse, Millipore, USA), GluR2/3 (rabbit, Millipore, USA) and β-actin (rabbit, Proteintech, USA). The blots were imaged and quantified using a Fusion Imaging System. The quantitative densitometric values of the proteins were normalized to that of GAPDH.

HLECs transiently transfected with plasmids or stable HEK-293 cells lines were harvested, and total protein was extracted using a RIPA protein extraction kit (Beyotime, China). Proteins were separated by 12% SDS-polyacrylamide gels and individually transferred to methanol-activated polyvinylidene difluoride membranes (Millipore, USA). The membranes were immunoblotted with a 1:1000 dilution of anti-Flag mouse monoclonal antibody (Sigma, CAS No. f1804, USA) or a 1:200 dilution of anti-Cx43 rabbit polyclonal antibody (Santa Cruz, CAS No. sc-9059, USA). A 1:1000 dilution of anti-GAPDH rabbit antibody (Diagbio, CAS No.db106, China) was used as an internal control. The fluorescent secondary goat anti-mouse (LI-COR Biosciences, CAS No.926-68072, USA) or goat anti-rabbit (LI-COR Biosciences, CAS No.925-32211, USA) antibodies (1:5000) were used for visualization. The signals were analyzed using the Image J program. The target protein expression levels were normalized relative to GAPDH expression. Each experiment was repeated at least three times.

Total cell protein was extracted from cells infected by GAS or E. coli using the RIPA protein extraction kit (Beyotime, CHN) and was then used for Western blotting to calculate the changes in A20, TRAF6, and p-P65. In brief, cells infected by GAS or E. coli were washed twice, mixed with RIPA lysis buffer, and then centrifuged at 8,000 g for 5 min at 4°C. Then, the supernatant was collected and protein concentration was determined according to the Bradford method (Bradford, 1976 (link)). Total cell lysates or M protein were then separated using sodium dodecyl sulfate polyacrylamide gel electropheresis (SDS-PAGE) and transferred to polyvinylidene difluoride (PVDF) membrane. After blocking, blots were incubated with rabbit monoclonal antibodies against A20/TNFAIP3 (Cell signaling Inc.), TRAF6 (Cell signaling Inc. USA), and p-NF-κB p65 (Cell signaling Inc. USA); β-actin (Cell signaling Inc.) was used as an internal control. Finally, blots were hybridized with horseradish-peroxidase-(HRP-)-conjugated goat anti-rabbit immunoglobulin G (IgG), incubated with enhanced chemiluminescence (ECL) solution (PerkinElmer Life Sciences, USA), and finally exposed to X-ray film.