Anti p p65 3033

After the brain sections were incubated with EDTA (pH 9.0) and heated in a water bath, they were treated with H₂O₂ and blocked with 10% bovine serum albumin (NA8692; Bomei Biotechnology, Hefei, China). Subsequently, the sections were incubated at 4°C overnight with a primary antibody (anti-p-p65, 3033; Cell Signaling Technology). Next, the sections were incubated using a Rabbit Two-step Detection Kit (PV-9001; ZSGB-BIO, Beijing, China). Finally, the sections were visualized using diaminobenzidine (ZLI-9018; ZSGB-BIO) and hematoxylin was used to label the nuclei.

LPS (L4391-1 MG), IL-1β (SRP3083), and TNFα (SRP2102) were purchased from Sigma‒Aldrich (St. Louis, MO). Anti-IKKα/b (2682), anti-p-IKKα (2078), anti-IκBα (9242), anti-p-IκBα (9246), anti-p65 (8242), anti-A20 (5630), and anti-p-p65 (3033) antibodies were purchased from Cell Signaling Technology (Danvers, MA, USA). Anti-Tubulin (T5168) and anti-OTUD4 (HAP036623) were purchased from Sigma‒Aldrich. Anti-MyD88 (sc-74532), anti-TLR4 (sc-293072), and anti-IRAK-1 (sc-5288) antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). The anti-RNF115/BCA2 antibody (ab187642) was purchased from Abcam (Cambridge, MA). Cells were harvested, lysed in RIPA lysis buffer supplemented with 1×Proteinase Inhibitor Cocktail (MedChemExpress, Monmouth Junction, NJ, USA, HY-K0010) and quantified using a BCA kit (Thermo Fisher). Briefly, ~40 µg of each protein sample was subjected to SDS‒PAGE and transferred to polyvinylidene fluoride (PVDF) membranes (Merck Millipore, Germany, #IPFL00010). The membrane was blocked with 5% milk for one hour and then incubated with primary antibody overnight at 4 °C. The membrane was incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies (Invitrogen, #31460 & #31430) for 1 hour at RT. Chemiluminescence was detected using an ImageQuant LAS4000 biomolecular imager (GE, USA) with Western HRP substrate (US Everbright, S6009 L).