Bm chemiluminescence western blotting kit

Extracts of cells or tissues were prepared. The protein concentration was determined by BCA assay kit (Thermo Scientific). Equal amounts of proteins (30 μg) were subjected to SDS-PAGE and transferred to polyvinylidene fluoride membranes (Millipore). The membranes were treated with 1% blocking solution in TBS for 1 h, and immunoblots were probed with the indicated antibodies: USP22 (Santa Cruz), BMI1 (Abcam), CD133 (Abcam), SOX2 (Abcam), P21 (Proteintech), ubH2A (CST), EZH2 (CST), H3K27Me3 (CST), tubulin (Santa Cruz), HA (Abmart) GAPDH (Santa Cruz), and actin (Santa Cruz) at 4°C overnight. Then the membranes were washed and incubated with HRP-labelled secondary antibodies (1:5,000; Santa Cruz). The fluorescence signals were detected by a BM Chemiluminescence Western Blotting kit (Roche). Densitometry quantification was calculated and analysed using ImageJ software.

Mice hippocampi were homogenized in lysis buffer consisting of TRIS-HCl, SDS, DTT, glycerol, and NP40. The homogenates were then centrifuged at 15,000×g for 10 min at 4 °C, and the supernatants were used for SDS-PAGE. Ten micrograms of protein was resolved on 10% SDS-PAGE gel and moved onto polyvinylidene difluoride (PVDF) (Millipore (Germany)) membranes. Membranes were blocked for 120 min with 5% non-fat skimmed milk and incubated with the following primary antibodies overnight: TLR4, pNF-κB (p65), NF-κB, IDO1, GAPDH, and β-actin. All antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Membranes were then washed 3 times with TBST (TBS+ tween 80) and incubated for 1 h at room temperature with secondary antibodies. Bands were visualized using the BM Chemiluminescence Western Blotting Kit acquired from Roche Diagnostics GmbH (Mannheim, Germany) and were detected using a gel documentation system. An open-source image-processing program, ImageJ, was used to quantify the optical densities of each band. The relative expressions of TLR4 and pNF-κB/total NF-κB were calculated and compared to the β-actin (TLR4 and pNF-κB/total NF-κB) or GAPDH (IDO1) as well as the control group.

Cell lysates were prepared using high KCl lysis buffer containing 10 mMTris-HCl, pH 8.0, 140 mMNaCl, 300 mMKCl, 1 mM EDTA, 0.5% TritonX-100 and 0.5% sodium deoxycholate with complete protease inhibitor cocktail (Roche) and 20 mM N-ethylmaleimide (NEMI) to block free thiol groups. Equal amounts of proteins (30 μg) were subjected to SDS-PAGE with loading buffer with or without β-mercaptoethanol and transferred to polyvinylidene fluoride (PVDF) membranes (Roche). The membranes were treated with 1% blocking solution in TBS for 1 h, immunoblots were probed with the indicated antibodies: anti-GFP (1:5,000; AbMart, Shanghai, China), anti-Flag (1:5,000; AbMart, Shanghai, China), anti-HA (1:5,000; AbMart, Shanghai, China), anti-Prdx-SO₃ (1:2,000; Abcam) at 4 °C overnight. Then the membranes were washed and incubated with POD-labeled secondary antibodies (1:125,000; Roche). The immunolabeled proteins were detected by BM Chemiluminescence Western Blotting kit (Roche).