Myd88

FFXHL pills (SFDA approval number Z11020012) was purchased from Beijing Tongrentang Co.,Ltd.Tongrentang Pharmaceutical Factory. Bovine type II collagen (20022), Incomplete Freund’s Adjuvant (IFA) (7002) were bought from Chondrex, Inc. (United States). Dexamethasone was purchased from Guangdong South China Pharmaceutical Group Co., Ltd. (Guangdong, China). Rheumatoid fibroblast-like synoviocyte transformed with SV40 T antigen (MH7A) was obtained from Jennio Biological Technology (Guangzhou, China). RPMI 1640 culture medium (22400071), trypsin, Pencillin-Streptomycin (P/S), and fetal bovine serum (FBS) were bought from Gibco (United States). Enzyme-linked immunosorbent assay (ELISA) were purchased from Sinouk Bio (Bei Jing, China) and Lianke Bio (Hang Zhou, China). TNF-a protein was obtained from PRPTO TECH (United States). TLR4, Myd88, p65, p-p65, IKKα/β, p-JNK1/2/3, JNK1/2/3, were purchased from Abcam (United Kingdom). p-Erk1/2, Erk1/2, p-p38, p38, β-actin, p-IKKα/β, p-IκBα, IκBα, p-Akt, Akt were purchased from CST (United States).

Immuno-blotting for protein analysis has been described earlier [25 (link)]. Following protein separation through a gradient polyacrylamide gel (4–12%) and electro-blotting onto a PVDF membrane, proteins were identified and their expression rate determined by Western-blotting. The following proteins were detected: β-defensin-1, C1qR, ICAM1, ICOS, ICOSL, MYD88, and SOCS (all Abcam, Cambridge, UK). Details of dilution and producers are provided in Table 2.
Membranes were blocked for 1 hour (room temperature) in PBS containing 0.01% Tween-20 and 2% bovine serum albumin. The primary antibodies were added at concentrations indicated in Table 1 and incubated overnight at 4°C. Following 3 washes with blocking buffer, membranes were incubated with secondary species specific antibodies labelled with horse radish peroxidase for 1 hour. Unbound antibody was washed off by 3 washes with blocking buffer and protein bands were visualised by exposure to X-ray films.
GAPDH expression was used to normalize protein expression and estimated changes induced by the different treatments.

RIPA buffer containing protease inhibitors was used to extract proteins from rat spinal cord tissues and BV-2 cells. Protein concentrations were then determined using a BCA Protein Assay Kit (Abcam). Protein samples (20 μg/lane) were separated via 10% SDS-PAGE and the resolved proteins were transferred onto PVDF membranes. Membranes were blocked with 5% bovine serum albumin at room temperature. After blocking, membranes were incubated overnight at 4 °C with primary antibodies against TLR4 (1:1000; Abcam), MyD88 (1:1000; Abcam), p65 (NF-κB) (1:1000; Abcam), p-p65 (phospho-NF-κB) (1:1000; Cell Signaling), NLRP3 (1:1000; Abcam), ASC (1:1000; Affinity Biosciences), caspase 1 (1:1000; Affinity Biosciences), N-GSDMD (1:1000; Abcam), and GAPDH (1:1000; Abcam). Thereafter, they were washed three times with Tris-buffered saline Tween-20. Subsequently, an HRP-conjugated IgG secondary antibody (1:5000; Santa Cruz, Waltham, MA, USA) was added and membranes were incubated at room temperature for 1 h. GAPDH was used as the internal reference. An enhanced chemiluminescence detection kit (Thermo Fisher Scientific) was used to detect the bands, which were then quantified using Gel-Pro Analyzer software (version 4.0; Media Cybernetics, Silver Spring, MD, USA).

The total protein was extracted from brain tissues, then homogenized in RIPA lysis buffer including protease inhibitor (Solarbio, China) and phosphatase inhibitor (Solarbio, China) for 30 min on ice. Protein of supernatant was collected after centrifugation for 15 min (13,600 ×g, 4°C). Total protein was detected using a bicinchoninic acid (BCA) kit (Thermo Scientific, USA). Brain lysates were electrophoresed on 10% SDS-PAGE, transferred to nitrocellulose membrane, then blocked with tris-buffered saline with Tween 20 (TBST) solution containing 3%BSA. Membranes were probed with primary antibodies for MyD88 (Abcam, USA), TRAF6 (Abcam, USA), NF-κB p65 (Abcam), Nrf2 (Bioss, China), HO-1 (Bioss, China), GAPDH (Abcam) and β-actin (Abcam) overnight at 4°C, and washed with TBST 3 times for 10 min. Membranes were treated with HRP-conjugated anti-rabbit antibody (Bioss, China) the next day. Protein bands were analyzed using enhanced chemiluminescence and calculated using ImageJ software.