Anti p65

Marc-145 cells cultured in 10-cm-diameter dishes were collected and lysed in nondenaturing lysis buffer (Sangon, Shanghai, China) supplemented with protease and phosphatase inhibitor cocktails. An equal mass of lysate was incubated overnight with 2 μg of anti-ARID3A (A7668, Abclonal, Wuhan, China), anti-p65 (A11201, Abclonal, Wuhan, China) or anti-IgG (Beyotime, Jiangsu, China) together with 25 μl of Protein A+G Agarose beads (Beyotime, Jiangsu, China). After centrifugation, the beads were washed with 1 mL of lysate buffer three or four times. Then, 15 μl of 2×SDS loading buffer was added to the beads, boiled for 10 min, and loaded onto an SDS–PAGE gel for western blot analysis.

The fruiting body of Hericium erinaceus was purchased from Jilin Jiaohe Songshan Food Co., Ltd. (Jiaohe, China). Dextran standards (670, 410, 270, 80, 25, 12 and 5 kDa) were purchased from Sigma Chemical Co. (St. Louis, MO, USA). Monosaccharide standards, including arabinose, mannose, fucose, xylose, rhamnose, galactose and glucose were purchased from Sigma Chemical Co. (St. Louis, MO, USA). Cell Counting Kit-8 was obtained from APExBIO Technology LLC (Houston, TX, USA). Neutral red staining solution, nitric oxide assay kit and BCA kit were obtained from Beyotime Biological Technology Co., Ltd. (Shanghai, China). Western Blot assay kit, SDS-PAGE kit, ELISA kits of TNF-α, IL-6, IL-10 and IFN were obtained from Boster Biological Technology Co., Ltd. (Wuhan, China). Anti-ERK, anti-phospho ERK, anti-JNK, anti-phospho JNK, anti-p38 MAPK, anti-phospho p38 MAPK, anti-Akt, anti-phospho Akt, anti-IkBα, anti-p65, anti-β-actin and anti-Histone H2A were purchased from ABclonal Biotechnology Co., Ltd. (Wuhan, China). Inhibitors of BAY11-7082, SB203580, SP600125, PD98059 and LY294002 were purchased from Selleck Chemicals (Shanghai, China).

Stimulated NP cells were collected and then lysed with RIPA lysis buffer (CWBIO, Beijing, China) for total protein, or with an extraction kit for cytoplasmic and nuclear protein (CWBIO). After being centrifuged and collected, the supernatant containing proteins was then quantified using a BCA assay kit (CWBIO). Afterwards, the proteins were separated by SDS-PAGE and electro-transferred to polyvinylidene difluoride membranes (Millipore, Darmstadt, Germany). After being blocked with 5% bovine serum albumin in TBST for 1 h, the membranes were then incubated at 4°C overnight with anti-CBX4 (1:1000; Abcam), anti-MMP3 (1:1000; Abcam), anti-MMP13 (1:1000; Abcam), anti-ADAMTS5 (1:1000; Abcam), anti-COX2 (1:1000; Abcam), anti-COL2A1 (1:1000; Abcam), anti-P53 (1:1000; Abcam), anti-P21 (1:1000; Abcam), anti-β-actin (1:1000; Abclonal, Wuhan, China), anti-p-p65 (1:1000; Abclonal), anti-p65 (1:1000; Abclonal), or anti-Histone H3 (1:1000; CST, Danvers, USA) antibodies. Afterwards, the membranes were incubated with the corresponding HRP-conjugated secondary antibodies (1:8000; Abclonal). The protein bands were visualized using an Enhanced Chemiluminescence kit (Vazyme, Nanjing, China) and then quantified by Image J (National Institutes of Health, Bethesda, USA).

Cells were lysed in radioimmunoprecipitation assay (RIPA) lysis buffer (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, NP-40, 1 mM EDTA, and 1× protease inhibitor cocktail [Roche, Mannheim, Germany]) according to the manufacturer’s protocol. The samples were separated with sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis, transferred onto nitrocellulose membrane, and probed with the indicated antibodies. Where indicated, Western blot signals were quantified by densitometry as previously described (51 (link), 52 (link)). The following antibodies were used: anti-polyclonal-NP (produced in our lab), anti-phospho-IRF3 (Abcam, Cambridge, UK), anti-IRF3 (Proteintech, Wuhan, China), anti-STAT1 (Abcam), anti-phospho-STAT1 (Abcam), anti-β-actin (TranGens Biotech, Beijing, China), anti-p65 (Abclonal, Wuhan, China) and anti-phospho-p65 (Abclonal).

Western blotting was performed as previously described^{50 (link)}. The primary antibodies are used as follows: anti-β-actin (Proteintech, Wuhan, China, 60008-1-Ig), anti-Lamin B1 (Proteintech, 12987-1-AP), anti-SLC7A2 (Abcam, ab140831), anti-p-P65 (Abcam, ab86299), anti-P65 (Abclonal, Wuhan, China, A19653), anti-p50 (Abclonal, A6667), anti-p-IKBα (Abclonal, AP0614), anti-p-AKT (Cell signaling technology, MA, USA, #4060), anti-AKT (Cell signaling technology, #4685), anti-p-JNK (Cell Signaling Technology, #9255), anti-JNK (Cell signaling technology, #9252), anti-p-ERK (Cell signaling technology, #4370), anti-ERK (Cell Signaling Technology, #4695), anti-p-P38 (Cell Signaling Technology, #4511), anti-P38 (Cell Signaling Technology, #8690), anti-p-stat3 (Tyr705) (Cell Signaling Technology, #9145), anti-stat3 (Tyr705) (Cell Signaling Technology, #4904), G9a (EHMT2) (Abcam, ab185050), H3K9me2 (Cell Signaling Technology, #4658), Twist1 (Cell Signaling Technology, #69366), Vimentin (Cell Signaling Technology, #5741), E-cadherin (Cell Signaling Technology, #3195), N-cadherin (Cell Signaling Technology, #13116), MMP9 (Cell Signaling Technology, #13667).