Sc 293072

Serial 4-μm-thick paraffin sections from CIA synovial tissues were deparaffinized in xylene and rehydrated through a graded ethanol series. Then, the sections were immersed in the 0.01 M citrate buffer (pH 6.0), and microwave irradiation was performed three times (8 min/time) for antigen retrieval. The sections were incubated in 5% goat serum for 1 h before immunostaining. In double-labeling experiments, the sections were incubated with anti-CD68 (pAb, 1:200, ab125212, Abcam, British; mAb, 1:100, NBP2-32831, NOVUS, USA), anti-Vimentin mAb (1:200, ab92547, Abcam, British; 1 : 200, ab20346, Abcam, British), anti-TLR2 pAb (1:150, 17236-1-AP, Proteintech, China), and anti-TLR4 mAb (1 : 100, sc-293072, Santa Cruz, USA) at 4°C overnight, followed by incubated with secondary antibodies, goat anti-rabbit IgG H&L(FITC) (1 : 200, ab6717, Abcam, British) or goat anti-mouse IgG H&L (Cy3) (1:200, ab97035, Abcam, British). The poststaining sections were examined with a full-spectrum scanning confocal microscope (FV1200, Olympus, Japan), and pairs of images were superimposed for colocalization analysis.

The antibody against TLR4 (#sc-293072) was from Santa Cruz Biotechnology (Santa Cruz, CA, USA). The antibodies against c-Jun NH₂-terminal kinase (JNK) (#9252S), p-JNK (#9255S) and Caspase-8 (#9746S), anti-rabbit IgG HRP-linked antibody (#7074S), and anti-mouse IgG HRP-linked antibody (#7076S) were from Cell Signaling Technology (Danvers, MA, USA). The antibody against GSDMD (#ab219800) was from Abcam (Cambridge, UK). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) antibody (#MB001) was from Bioworld Technology (Qixia District, Nanjing, China).
Western blotting was performed as we have previously described [64 (link)]. The proteins were transferred to polyvinylidene fluoride membranes (Millipore, Bedford, MA, USA), which were incubated with primary and secondary antibodies by standard techniques. The enhanced chemiluminescence (ChemiDoc XRS + System, Bio-Rad, Hercules, CA, USA) was used to accomplish immunodetection.

Primary amniocyte and AV3 cells were lysed in a RIPA buffer (20 mM Tris-HCl pH = 7.5, 150 mM NaCl, 1% Nonidet P40, 0.5% sodium deoxycholate, 1 mM EDTA, 0.1% SDS, and 1× Complete Protease inhibitor cocktail (Roche)) for 30 min at 4°C. The amnion and choriodecidua samples were homogenisated, as previously described (Choltus et al., 2020 (link)). The protein concentration of the supernatant was determined using a Pierce BCA Assay Kit (Pierce). Forty µg of denaturated proteins were subjected to a Western blot analysis after 4–15% MiniPROTEAN TGX StainFree gel electrophoresis (Bio-Rad), which was followed by probing antibodies against TLR4 or β-Actin (TLR4:1:400, Sc-293072, Santa-Cruz Biotechnology, β-Actin:1:10,000, MA1-91399, Thermo Fisher Scientific). The signal was detected with a peroxidase-labelled antimouse antibody at 1:10,000 (Sc-2005, Santa-Cruz Biotechnology) and visualised with ECL or ECL2 Western blotting substrate (Pierce) using a ChemiDoc MP Imaging System and Image Lab software (Bio-Rad). The results were obtained from at least seven independent experiments, and the relative TLR4 ratio was expressed as a function of β-Actin or total protein loaded by well.

Cell lysate proteins (30 μg) were separated by SDS-PAGE and transferred to PVDF membranes. Membranes were first blocked in 5% fat-free milk in PBST (Phosphate buffer saline, 0.1% Tween 20) and then probed with primary antibodies in 1% fat-free milk in PBST. The following antibodies were used with the indicated dilution: anti-CASP1 (1:1,000, Adipogen AG-20B-0042), anti-CD36 (1:1,000, R&D Systems AF2519), anti-FCGR2B (1:1,000, Cell Signaling 96397), anti-IL1B (1:1,000, GeneTex GTX 74034), anti-MRC1 (1:1,000, Abcam ab64693), anti-NLRP3 (1:3,000, Adipogene AG-20B-0014), anti-TLR2 (1:500, R&D Systems AF1530), and anti-TLR4 (1:1,000, Santa Cruz sc-293072). Membranes were washed in PBST and incubated with the appropriate HRP-conjugated secondary antibodies (1:5,000) in 1% fat-free milk in PBST. After five washes, immunoreactivity was revealed by incubating membranes with the HRP SuperSignal West Femto Maximum Sensitivity Substrate (ThermoFisher Scientific), and the signal was detected by the Chemidoc XRS system (Bio-Rad). Protein amount loading control and normalization were achieved by probing the membranes with α-tubulin (TUBA1A) (1:4,000, Sigma-Merck T5168) or β-actin (ACTB) (1:10,000, Sigma-Merck A5441) antibodies.

Where indicated we used an anti-TLR4 monoclonal antibody (sc-293072 Santa Cruz Biotechnologies, Dallas, TX, USA) and a goat anti-mouse alkaline-phosphatase conjugated antibody as secondary antibody (sc-2008 Santa Cruz Biotechnolgies, Dallas, TX, USA).

The following antibodies were purchased from Cell Signaling Technology (Danvers, MA), p‐IRS1 (Ser307; #2381; 1:1000), IRS1 (#2382; 1:1000), p‐AKT (Ser473; #4060; 1:1000), AKT (#4691; 1:1000), p‐mTOR (Ser2448; #5536; 1:1000), mTOR (#2983; 1:1000), p‐p65 (#3033; 1:500), p65 (#8242; 1:500), p‐GSK3β (Ser9; #5558; 1:1000), GSK3β (#12 456; 1:1000), FOXO1 (#2880; 1:1000), VAMP2 (#13 508; 1:250), VAMP3 (#13 640; 1:1500), VAMP8 (#13 060; 1:500), GADPH (#97 166; 1:4000), HA‐Tag (#3724; 1:1000), Flag‐Tag (#14 793; 1:1000), HRP‐linked anti‐mouse IgG (#7076; 1:1000), and HRP‐linked antirabbit IgG (#7074; 1:1000). Antibodies against TLR4 (sc‐293072; 1:1000), SNAP23 (sc‐166244; 1:1000), syntaxin 4 (sc‐101301; 1:500), and Na⁺/K⁺‐ATPase (sc‐58629; 1:1000) were obtained from Santa Cruz Biotechnology (Dallas, TX). p‐IRS1 antibody (Tyr608; 09–432; 1:1000) was purchased from Millipore (Burlington, MA). Antibodies against TMEM16A (ab53212; 1:2000 for western blotting, 1:100 for immunohistochemistry and immunofluorescence), HNF4 (ab41898; 1:100), CD68 (ab125212; 1:50), p‐FOXO1 (Ser256; ab131339; 1:1000), and GLUT2 (ab54460; 1:500) were purchased from Abcam (Cambridge, UK). Unless otherwise indicated, all chemicals were purchased form Sigma‐Aldrich (St. Louis, MO).