Ab2064

Cells or tissues were harvested at indicated times and homogenized in ice-cold suspension buffer supplemented with a proteinase inhibitor cocktail (Sigma-Aldrich). Protein concentrations were determined using the BCA Protein assay kit (Thermo Scientific, Waltham, MA). Equal amounts of protein were fractionated by SDS polyacrylamide gels, followed by immunoblotting with the following primary antibodies: NF-κB Pathway Sampler Kit (diluted at 1:1000, 9936, CST, MA), phospho-MAPK Family Antibody Sampler Kit (diluted at 1:1000, 9910, CST, MA), MAPK Family Antibody Sampler Kit (diluted at 1:1000, 9926, CST, MA), phospho-Stat6 antibody (Rabbit polyclonal, diluted at 1:1000, ab28829, Abcam), Stat6 antibody (Rabbit polyclonal, diluted at 1:1000, ab44718, Abcam), TRAF6 antibody (Rabbit polyclonal, diluted at 1:1000, ab94720, Abcam), TLR4 antibody (Rabbit polyclonal, diluted at 1:1000, ab13556, Abcam), CD14 antibody (Rabbit polyclonal, diluted at 1:1000, ab203294, Abcam), Myd88 antibody (Rabbit polyclonal, diluted at 1:1000, ab2064, Abcam), PPARγ antibody (Rabbit monoclonal (C26H12), diluted at 1:1000, sc-7273, Santa Cruz Biotechnology). Membranes were then incubated with peroxidase-conjugated secondary antibody, and specific bands were detected with a Bio-Rad (Hercules, CA) imaging system. Original blots were provided in Supplementary Fig. 8.

Total protein was extracted using radioimmunoprecipitation assay (RIPA) lysis buffer (Boster Biological Technology Co. Ltd.) containing protease inhibitors. The protein concentration was determined using a bicinchoninic acid (BCA) protein assay kit (Boster Biological Technology Co. Ltd.). Next, the protein was separated by 10% sodium dodecyl sulphate‐polyacrylamide gel electrophoresis (SDS‐PAGE) and transferred onto the poly(vinylidene fluoride) membranes. The membrane was then blocked with 5% bovine serum albumin (BSA) at room temperature for 2 hours to avoid non‐specific binding, followed by overnight incubation with the following primary rabbit polyclonal antibodies (Abcam Inc) to TLR2 (ab213676, 1:500), TLR4 (ab13556, 1:500), MYD88 (ab2064, 1:1000), TGF‐β (ab92486, 1:500), MMP9 (ab38898, 1:500), TIMP‐1 (ab61224, 1:1000) and GAPDH (ab181602, 1:5000) at 4°C. Afterwards, horseradish peroxidase‐conjugated secondary antibody goat anti‐mouse (ab205719, 1:2000, Abcam Inc) was added to the membrane and incubated for 1 hour at room temperature. The membrane was visualized using an enhanced chemiluminescence (ECL) kit (EMD Millipore). The greyscale quantification of bands in Western blot images was performed using Image J analysis software with GAPDH as an internal reference.16

SCC13 TLR4 overexpressing and SCC13 control cells were grown in 6 cm dishes in serum free DMEM medium for 24 hours. Afterwards cells were stimulated with ultrapure LPS (10μg/ml, Sigma) for different time intervals (0–60 min). Cells were lysed in RIPA buffer; the lysates were collected after each time interval and subjected to SDS-PAGE, followed by immunoblotting. The phosphorylated and non-phosphorylated form of ERK1/2 and JNK was detected by using specific rabbit polyclonal antibodies (anti-pp42/44 (#9101), anti-p42/44 (#9102); anti-rabbit HRP (#7074), all from Cell Signaling; anti-β-Actin (sc-47778, Santa Cruz Biotechnology, anti-mouse HRP (ab6728, Abcam). The expression of MyD88 (ab2064, Abcam) and IRAK-1 (D51G7, #4504, Cell signaling) upon LPS stimulation was analyzed using the same protein lysates. The secondary HRP-conjugated antibodies were used based on their species specificity to the primary antibodies.
ELISA: Cells were grown in 24 well plates till they reach 60% density. The expression of secreted IL-6 was analysed by ELISA (R&D Systems) before and after LPS stimulation at 6h and 24 hours. As controls unstimulated TLR4+ and TLR4- cells were used.

The cell lysates were prepared with RIPA buffer containing protease inhibitors (Sigma-Aldrich). The membranes were incubated overnight at 4 °C with of the primary antibodies at a dilution of 1:1000. A secondary antibody was then used for immunostaining for 1 h at room temperature. The primary antibodies used in this study are anti-MyD88 (Abcam, ab2064), anti-CBP (Abcam, ab50702), anti-SP1 (Abcam, ab124804) anti-NF-κB p65 (Abcam, ab16502), and anti-β-actin (Abcam, ab6276) antibody.

Adipose tissues were homogenized in ice-cold lysis buffer. After homogenization for 30 min on ice, the tissue homogenates were centrifuged at 13,000 r/min for 30 min at 4 °C. The bicinchoninic acid method was applied to determine the total protein concentration. Equivalent amounts of proteins were loaded in each lane, denatured, and then frozen at − 80 °C before western blotting. The proteins were subsequently subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis and transferred onto polyvinylidene fluoride membranes. After incubation with the apposite primary antibody at 4 °C overnight and secondary antibodies at room temperature for 2 h, polyvinylidene fluoride membranes were washed 3 times in PBS for 10 min, and the targeted proteins were detected by using enhanced chemifluorescence reagent.
The membranes were incubated with antibodies against TLR4 (ab13556, Abcam), IRS-1 (ab131487, Abcam), MyD88 (ab2064, Abcam), NF-κB (8242S, CST), andphospho-NF-κB (3033, CST). All the samples were analysed in an average of 3 independent tests by using different gels. Bands were quantified by ImageJ.

Western blot analyses were performed using an anti-alpha V antibody (ab124968, and ab112487 Abcam, Cambridge, MA), phospho-FAK antibody (ab24781, Abcam, Cambridge, MA) FAK antibody (ab131435, Abcam, Cambridge, MA), TLR2 antibody (ab213676, Abcam, Cambridge, MA), TLR4 antibody (SC-293072, Santa Cruz Biotecnology, Santa Cruz, CA), or MyD88 antibody (ab2064, Abcam, Cambridge, MA) followed by a horseradish peroxidase-conjugated anti-mouse antibody (SC-2005, Santa Cruz Biotechnology). Blots were then developed with the ECL-plus detection system (Thermo Fisher Scientific, Waltham, MA). To evaluate the samples for equal protein loading, membranes were stripped and re-probed with an anti-β-actin antibody (SC-1615, Santa Cruz Biotechnology).

Total protein was extracted using RPPA lysis buffer (R0010, Solarbio) containing PMSF, and protein concentration was determined using a BCA kit (Thermo, USA). The sample was denatured by heating in the loading buffer at 100°C for 10 min. Then, 50 μg of protein was loaded, electrophoresed at 70 V for 3h, and transferred onto a PVDF membrane (ISEQ00010, Millipore, Billerica, MA, USA) at a constant flow of 150 mA. The membrane was blocked with 5% skim milk (Shanghai Xinyu Biotechnology Co., Ltd.) at room temperature for 4h at 20°C; washed with TBST; incubated with rabbit antibodies of TLR4 (ab13556, 1:500, Abcam, UK), MyD88 (ab2064, 1:500, Abcam, UK), NF-κB p50 (ab220803, 1 µg/mL, Abcam, UK), p-NF-κB p50 (phospho S337) (ab28849, 1 μg/mL, Abcam, UK), and GAPDH (ab22555, 1:2,000, Abcam, UK) overnight at 4°C; and washed with TBST again. Subsequently, the membrane was incubated with HRP-labeled goat anti-rabbit IgG antibody (Beijing Zhongshan Biotechnology Co., Ltd., diluted 1:5,000) for 2h, washed with TBST, and developed using the ECL fluorescence detection kit (Cat. No. BB-3501, Amersham, UK) on a Bio-Rad image analysis system (BIO-RAD, USA).