Anti p65

The hippocampus was separated from the brain tissue and homogenized and lysed by adding a protein extraction solution (PRO-PREP, iNtRON, Sungnam, South Korea). After measuring the total protein concentration using Bradford reagent (Bio-Rad, Hercules, CA, USA), western blotting was performed as previously described (Han et al., 2019 (link)). Protein-specific primary antibodies were purchased from Novus Biologicals (anti-APP; Littleton, CO, USA), Santa Cruz Biotechnology (anti-GFAP and anti-β-actin; Dallas, TX, USA), Cell Signaling Technology (anti-iNOS, anti-COX-2, anti-p65, and anti-p50; Danvers, MA, USA), and Abcam [anti-BACE1, anti-ionized calcium-binding adapter molecule 1 (IBA-1), and anti-Foxp3; Cambridge, MA, USA]. Secondary antibodies were purchased from Santa Cruz Biotechnology (anti-mouse, anti-rabbit, and anti-goat).

Cells were lysed by RIPA lysis buffer (Beyotime, China). After centrifugation, 50 μg of protein lysate were pretreated with 20 μl protein A/G agarose (Santa Cruz, USA) and incubated for 1 h at 4 °C with gentle rotation. Lysates were incubated with 1 μg anti-p65 (Abcam USA) at 4 °C overnight with gentle rotation. The next day, 80 μl protein A/G agarose was added, and the suspensions were incubated for 2 h at 4 °C with gentle rotation. Suspensions were centrifuged, and the agarose phase collected and washed three times with PBS. Samples were eluted with 30 μl protein lysis buffer (Beyotime, China). After elution, loading buffer (7.5 μl of a 5x solution; Beyotime, China) was added, and samples were boiled for 5 min. For Western blotting, anti-wnt1 or anti-SRA antibody (1:1000, CST/millpore, USA) was used to detect the protein.

The brain tissue samples were processed as described in previous report^{41 (link)}. Briefly, 30 μg of total protein from each sample was electrophoresed on 10% SDS-PAGE gel and transferred to PVDF membrane. After blocking, the membrane was incubated overnight with appropriate primary antibodies: anti-p65 (Abcam, 1:1000), anti-β-actin (Abcam1:50000), anti-IL-1alpha (Abcam, 1:200), anti-IL6 (Abbiotec, 1:500), anti- neurabin 2 (Abcam, 1:1000) followed by incubation with the secondary HRP-conjugated antibody i.e. goat anti-mouse HRP (Santacruz, 1:10000) and goat anti-rabbit HRP (Santacruz, 1:5000) at room temperature for 2 h.

Total protein was extracted using 2% SDS lysis buffer including protein phosphatase inhibitor (Applygen, Beijing) and heated for 10 min. Protein samples were separated by 8% (v/v) SDS-PAGE gels and transferred onto nitrocellulose membranes (Millipore, Ireland). After blocking in 5% (w/v) Albumin Bovine-V (BSA-V, Solarbio, China) for 1 h at room temperature, the protein bands were incubated with primary antibodies at a dilution ratio of 1:1000 overnight at 4 °C. The primary antibodies contain anti-GAPDH (FL-335; Santa Cruz Bio technology), anti-TGM2 (Proteintech), anti-PD-L1 (Proteintech), anti-STAT3 (CST) and anti-Phospho-STAT3 (Ser705, CST), anti-Akt (CST), and anti-Phospho-Akt (Ser473, CST), anti-P65 (Abcam) and anti-Phospho-P65 (Ser536, Abcam). The protein bands were incubated in HRP-conjugated secondary antibodies (Zsbio, China, 1:5000) at room temperature for 1 h.

Western blotting analysis was carried out as previously described [25 (link)]. The following antibodies were used: anti-Bcl-2 (#ab182858), anti-Bax (#ab182733), anti-Bcl-xl (#ab32370), anti-p-P65 (#ab76302), anti-P65 (#ab32536), anti-p-IκBα (#ab133462), anti-IκBα (#ab32518) (Abcam Cambridge, MA, USA), anti-FXR (#sc-25309), anti-TLR4 (#sc-293072) (Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-p-ERK1/2 (#4370), anti-ERK1/2 (#4695), anti-p-P38 (#4511), anti-P38 (#8690), anti-p-JNK1/2 (#4668), anti-JNK1/2 (#9252), anti-CCL2 (#66272-1-Ig), and anti-β-actin (#66009-1-Ig) (Proteintech, Wuhan, China).

Cells’ proteins were extracted by RIPA lysis buffer (Beyotime) and each sample was subjected to 10% SDS–PAGE. Then they were transferred onto PVDF membranes (Owl Scientific) and the membranes were incubated with blocking solution for 1 h. Anti-GAPDH (1:1,000; Abcam), anti-ABIN1 (1:1,000; Abcam), anti-p65 (1:1,000; Abcam), and anti-p-p65 (1:1,000; Abcam) were applied as primary antibodies. After washing with PBST for four times, membranes were incubated with secondary antibody for 1 h. The proteins were quantified using ECL detection reagent (Pierce Biotechnology).