Ab76429

FNT (formononetin) was purchased from TargetMol (Boston, MA, USA). 4-nitrophenyl N-acetyl-β-D-glucosaminide was purchased from Sigma (St. Louis, MO, USA). Dexamethasone (Dexa), ketotifen fumarate (Keto), Evans blue, and toluidine blue were purchased from Meilun Biotechnology (Dalian, China). Primary rabbit antibodies against IκBα (ab76429, monoclonal), p-IκBα (ab133462, monoclonal), p65 (ab32536, polyclonal), GAPDH (ab181602, monoclonal), and loricrine (ab176322, monoclonal) were purchased from Abcam (Cambridge, MA, USA). Antibodies targeting filaggrin (DF13853, monoclonal) were purchased from Affinity Biosciences (Cincinnati, OH, USA). Anti-p-p65 (sc-135769, monoclonal), anti-rabbit IgG-HRP (sc-2357, monoclonal), and 4% paraformaldehyde solution in PBS were from Santa Cruz Biotechnology (Dallas, TX, USA). 2,4-dinitrochlorobenzene (DNCB) was purchased from Tokyo Chemical Industry (Tokyo, Japan). C48/80 was purchased from Cayman Chemical (Ann Arbor, MI, USA).

Skin tissue was lysed with RIPA buffer, the protein concentration was measured by BCA protein assay, and equal protein amounts were added electrophoretically separated on precast 4%–20% Precast Gels (Meilun) and then transferred onto PVDF membranes. Following incubation with 5% nonfat milk for 2 h, the membranes were treated with primary antibodies overnight at 4°C. The primary antibodies were used as follows: RelB (10544S, CST, 1:1000), IκBα (ab76429, Abcam, 1:1000), and NKG2-D (sc-515599, Santa Cruz, 1:1000). After washing three times, the corresponding secondary antibody were added and incubated at room temperature for 2 h. Finally, the protein bands were visualized with a Gel Doc imaging system and analyzed with the ImageJ software.

A Nuclear and Cytoplasmic Extraction Reagents kit (78835, Thermo Fisher Scientific, USA) was used to extract cytoplasmic and nuclear proteins. A BCA Assay Kit (23223, Thermo Scientific, USA) was used to measure the protein concentration. In this experiment, the protein was electrophoresed on 10% SDS‒PAGE gels and then transferred to PVDF membranes (Millipore, Bedford, MA, USA). The membranes were coincubated overnight at 4 °C with specific primary antibodies (ab214429, ab32535, ab3778, ab41037, ab307674, ab32561, ab15580, ab133462, ab76429, ab124957, ab181602, ab32536, Abcam, Cambridge, UK) (PA5-61136, Invitrogen, USA) (18165-1-AP, Proteintech, China). The membranes were rewarmed for 2 hours and then incubated with rabbit IgG (ab97051, Abcam, Cambridge, UK) at 37 °C for 1 hour. The bands were detected using an ECL Western blot kit (32209, Thermo Fisher Scientific, USA) and ImageQuant™ LAS500 (Cytiva, China). In this study, GAPDH and Lamin B were used as controls. The methodology employed for the utilization of antibodies is documented in Supplementary Table 2.

Immunoblot assays were conducted as described previously [28 (link)]. The following primary antibodies were used: anti-IGFBP3 (1:1000, GTX113364, GeneTex, Irvine, CA, USA), anti-cytochrome c (1:1000, 556,433, BD Biosciences, Franklin Lakes, NJ, USA), anti-cleaved caspase 3 (1:500, IMG-144A, IMGENEX, San Diego, CA, USA), anti-light chain 3B (1:500, LC3B, GTX127375, GeneTex), anti-NF-κB (1:1000, #6956, Cell Signaling), anti-phosphorylated NF-κB (1:1000, #3033, Cell Signaling), anti-IκBα (1:1000, ab76429, Abcam, Cambridge, UK), anti-phosphorylated IκBα S32 (1:1000, ab92700, Abcam), and anti-α-tubulin (1:5000, GTX628802, GeneTex). Protein levels were determined by measuring the intensity of bands on western blots using ImageJ (National Institutes of Health, Bethesda, MD, USA). Protein levels were normalized against α-tubulin as an internal control. The relative ratio was calculated by dividing the normalized protein levels in stably expressing cells with that in control cells.

The liver was weighed and ground with liquid nitrogen. The milled tissue powder was added into lysis solution containing RIPA (tissue weight: volume of lysis solution 20 mg:150–250 μL) for lysis. BCA protein detection kit was used to detect protein concentration. The total protein (10 μg) of each sample was added to SDS-PAGE gel electrophoresis to separate the protein and transferred to PVDF membrane (Millipore). The PVDF membrane containing protein was incubated in a closed solution for 2 hours and then incubated in the required primary antibody, including TLR4 antibody (1 : 500, ab13556; Abcam), MyD88 antibody (1 : 1000, ab219413; Abcam), TRAF6 (1 : 1000, ab33915; Abcam), IκB antibody (1 : 2000, ab76429; Abcam), P-IκB antibody (1 : 1000, 2859 S; Abcam), NF-κB antibody (1 : 2000, ab16502; Abcam), and β-actin antibody (1 : 10000, 20536-1-AP; Proteintech), incubated at 4°C overnight. After incubating with HRP-conjugated AffiniPure Goat Anti-Rabbit IgG (H + L) (1 : 5000, SA00001-2, Proteintech) and HRP-conjugated AffiniPure Goat Anti-Mouse IgG (H + L) (1 : 5000, SA00002-1, Proteintech) for 1 h, the PVDF membrane was washed with solution TBST, treated with chemiluminescence reagent, taken photos by exposure, and performed quantitative analysis with Image-Pro Plus 6.0.

Total proteins of cells and mouse articular cartilage tissues were isolated with RIPA buffer containing 1% protease inhibitors (Sigma-Aldrich). Equal amount of each protein sample was loaded on 12.5% SDS-PAGE and then transferred to PVDF membranes (Millipore). Blocking was done by incubating the membranes with TBST containing 5% slim milk at 37 °C for 2 h and the corresponding primary antibodies at 4 °C for overnight. Membranes were then incubated with HRP-conjugated secondary antibodies (1:2000, Abcam) at RT for 1 h. Proteins were detected with an enhanced chemiluminescence (ECL) kit (Pierce). Primary antibodies used were anti-β-actin (1:1000, Abcam, ab8227), anti-p65 (1:1000, Abcam, ab16502), anti-IkBα (1:1000, Abcam, ab76429), and anti-HMGB1 (1:1000, Abcam, ab77302). Proteins were quantitatively analyzed and normalized relative to β-actin using Image-Pro Plus software (v6.0) (Media Cybernetics).