Mini protean tgx 4 20 gel

Samples diluted 1:400 in Laemmli buffer were separated on Mini-PROTEAN TGX 4-20% gels (4561096 or 4561094, Bio Rad) and blotted onto 0.22μm nitrocellulose membrane using the high MW program of the TransBlot Turbo system (Bio Rad). The membrane was blocked in 5 % skim milk in Tris Buffered Saline (TBS, Fischer scientific), incubated with 1:50,000 diluted horseradish peroxidase (HRP) conjugated goat anti-human IgG (H+L) (109-035-088, Jackson ImmunoResearch) and 1:50,000 HRP conjugated goat anti-human albumin (PA1-28334, Pierce). To detect C4 cleavage, the membrane was incubated with goat anti-human C4 serum (1:2,000, A205, CompTech) as primary antibody and with donkey IgG-HRP anti-goat-IgG (1:40,000, 6420-05, Southern Biotech) as secondary antibody. Blots were developed with Amersham ECL Prime solution reagent (GE Healthcare).

Bone marrow adherent cells were cultured as previously described. All cells were lysed in RIPA buffer (Sigma, St. Louis, MS, USA) supplemented with protease inhibitors (Roche, Indianapolis, IN, USA), centrifuged at 13,000× g for 20 min at 4 °C, and the supernatants were obtained. Protein concentration was determined in the clarified supernatant using Bio-Rad protein assay (Bio-Rad Laboratories, Hercules, CA, USA). Proteins were electrophoresed on Mini-PROTEAN TGX 4−20% gels (Bio-Rad Laboratories) and transferred to nitrocellulose 0.2 mm (GE Water & Process Technologies, Feasterville-Trevose, PA, USA). Membranes were blocked in 5% rehydrated non-fat milk in PBS-Tween 0.5% for 1 h at room temperature. Primary antibodies for PD-L1 (1:250) (Catalog#AF1019) (R&D Systems), NLRP3 (1:1000) (Catalog#AG-20B-0014-C100) (Adipogen, San Diego, CA, USA), and IL-1β (1:250) (Catalog#AF-401-NA) (R&D Systems) were used in combination with peroxidase-conjugated secondary antibodies. A primary antibody against β-actin (Santa Cruz Biotechnology, Dallas, TX, USA) was used to assess protein loading. Chemiluminescence was captured using Bio-Rad Chemidoc MP Imaging System (Bio-Rad Laboratories). Images were quantified using ImageJ (U.S. National Institutes of Health, Bethesda, MD, USA).

Flies laid on grape-agar plates for two hours and embryos were either aged two hours at room temperature or taken directly after collection. Embryos were dechorionated with bleach, rinsed, and frozen in aliquots of ~25 embryos at -80 C. Embryos were homogenized in 25 μl RIPA buffer (Sigma cat # R0278) supplemented with 1 mM DTT and protease inhibitors (Sigma cat # 4693116001) using a plastic pestle. After homogenization, samples were mixed with 25 μl 2x Laemmli buffer (Bio-Rad # 1610737EDU), boiled for 3 minutes, and spun at 21,000 x g for 1 minute. Samples were loaded onto Bio-Rad mini Protean TGX 4–20% gels (# 4561096) and run at 200V for 30 minutes. Protein was transferred at 350 mA for one hour to Immobilon PVDF membrane (Millipore-Sigma # IPVH00010). Blots were blocked for one hour in PBST (1x PBS with 0.1% Tween) with 5% nonfat milk, and stained with primary antibodies (courtesy of Maria Cristina Gambetta [27 (link)], 1:1000 in PBST with 3% BSA) for one hour. Blots were then washed 3 times for 3 minutes rotating in PBST and probed with an HRP-conjugated anti-Rabbit secondary antibody (Rockland Trueblot, # 18-8816-33, 1:1000 in PBST with 5% milk) for one hour. After extensive washing with PBST, blots were developed with Clarity ECL reagents (Bio-Rad # 1705060) and imaged. Validation of the loss of maternal dCTCF is shown in S1 Fig.

The protein concentration of the synovial tissue extracts (L) was determined in the clarified supernatants using a Bio-Rad protein assay (Bio-Rad Laboratories, Hercules, CA, USA). Proteins were electrophoresed on Mini-PROTEAN TGX 4–20% gels (Bio-Rad Laboratories) and transferred to nitrocellulose of 0.45 μm pore size (GE Healthcare Life Sciences, Marlborough, MA, USA). Membranes were blocked in 5% dried milk in PBS-Tween 0.5% for 1 hour at room temperature. Phosphorylated stress-activated protein kinase/c-Jun N-terminal kinase (JNK) (1:500; Cell Signaling Technology, Danvers, MA, USA), IL-1β (1:1000, AF-401; R&D Systems), and NLRP3 (1:1000, Cryo-2; AdipoGen Life Sciences, San Diego, CA, USA) were used as the primary antibodies. Peroxidase-conjugated secondary antibodies and chemiluminescence were used to develop the blots. A primary antibody against β-actin (Santa Cruz Biotechnology, Dallas, TX, USA) was used to assess protein loading.