Cytoplasmic protein extraction kit

Cells or ground tissues were dissolved in radio immunoprecipitation assay (RIPA) lysis buffer containing 1 mM protease inhibitor cocktail and 1 mM phenylmethanesulfonyl fluoride (PMSF). Cytoplasmic proteins were extracted using the cytoplasmic protein extraction kit (Beyotime Institute of Biotechnology, Haimen, China) according to the manufacturer’s instructions. Proteins (10 µg/lane) were separated by 8% SDS–PAGE and transferred onto polyvinylidene fluoride (PVDF) membranes via electroblotting. The membranes were blocked with 5% bovine serum albumin in phosphate buffer saline (PBS) for 1 hour and then incubated with antibodies against MUC16 (1:1,000), phosphoinositide 3-kinase (PI3K, 1:2,000), or β-actin (1:5,000) overnight at 4 °C. After washing in tris buffered saline tween (TBST, 50 mmol/L Tris, 145 mmol/L NaCl, and 0.05% Tween-20) three times for 10 minutes each, the membranes were incubated with secondary antibodies (1:5,000, Abcam) for 1 hour at room temperature. All experiments were repeated at least three times.

Liver tissue was used in Western blotting, and cytoplasmic protein was isolated using a Cytoplasmic Protein Extraction kit (Beyotime, Jiangsu, China). Protein concentration was measured using the bicinchoninic acid method (Beyotime, China), and an amount of protein (25 μg) was loaded into 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels. The membranes were incubated with anti-AMPK, anti-p-AMPK, and anti-GAPDH at 4°C overnight. After washing, the membranes were incubated with a secondary antibody (1: 5000; CST, USA). The signal was detected using an enhanced chemiluminescence detection kit (Amersham ECL RPN 2106 Kit, Amersham Pharmacia Biotech, QC, Canada).

For nuclear protein extraction in cells, nuclear and Cytoplasmic Protein Extraction Kit (Beyotime, Nanjing, China) was used according to the manufacturer's instructions. For extraction of total protein samples, cells and hepatic tissues were homogenized using 10% (wt/vol) hypotonic buffer (pH 8.0, 5 μg/ml soybean trypsin inhibitor, 1 mM EDTA, 4 mM benzamidine, 1 mM Pefabloc SC, 25 mM Tris-HCl, 5 μg/ml leupeptin, 50 μg/ml aprotinin) to yield a homogenate. The final supernatants were collected via centrifugation at 12,000 rpm for 15 min at 4 °C. Bicinchoninic acid (BCA) protein analysis kit (Thermo Fisher Scientific, USA) was used for the measurements of protein concentration according to its instructions. Then, 40 μg protein was subjected to 10–12% SDS-Polyacrylamide-Gel-Electrophoresis (SDS-PAGE) and transferred to polyvinyldene fluoride (PVDF) membranes (Millipore, USA). After blocking in 5% skim milk, all membranes were incubated with primary antibodies (Supplementary Table 2) overnight at 4 °C. Then, the membranes were incubated with corresponding secondary antibodies (Supplementary Table 2) for 1 h at room temperature. The signal was detected with the enhanced chemiluminescence (ECL) Detection system (Thermo Fisher Scientific). Each protein expression was analyzed using Image Lab Software (Version 1.4.2b, National Institutes of Health, USA) and normalized to GAPDH.

Cell lysate were acquired after harvested cells were treated with RIPA buffer system containing protease inhibitor cocktail and PMSF (phenylmethanesulfonyl fluoride) at 1 mmol/L (Santa Cruz). Cytoplasmic and nuclear protein samples were extracted by using a Cytoplasmic Protein Extraction kit (Beyotime) and a Nuclear Protein Extraction Kit (Beyotime). Protein concentration was determined by using a BCA Protein Assay Kit (Beyotime). After separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), proteins were then transferred to polyvinylidene fluoride membranes. After incubated with blocking buffer (Beyotime) for 1 hour, the membranes were probed by primary antibodies specific to RAGE (Abcam, 1:2500), TLR4 (CST, 1:2000), MyD88 (Abcam, 1:2500), MMP9 (Abcam, 1:2000), NFκB p65 (Abcam, 1:2000), IκBα (CST, 1:2500), GAPDH (Abcam, 1:5000) and Lamin B1 (Abcam, 1:2500) at 4℃ for 10 hours. After Tris-buffered saline Tween 20 (TBST) washing, horseradish peroxidase-conjugated secondary antibodies were used to incubate the membranes at room temperature for 20 min. ECL Chemiluminescent Substrate Reagent Kit (Invitrogen) was used to develop the membranes. Image of the immunobands were captured and analyzed by a luminescent image analyzer.

Nuclear protein was obtained by using a Cytoplasmic Protein Extraction Kit (Beyotime Biotechnology) per manufacturer's protocols.

BMMs were cultured with PDA, ALN, and PDA-ALN. After 7 days of incubation, cells were treated with Cytoplasmic Protein Extraction Kit supplemented with protease inhibitor cocktail and protein phosphatase inhibitor (Beyotime Biotechnology). The solution was separated by SDS-PAGE electrophoresis and transferred to PVDF membranes (Millipore, USA), which were incubated with 5% nonfat milk (CST, USA) in PBS for 1 h room temperature at first, then the primary antibody at 4 °C overnight. On the second day, the membranes were incubated with secondary antibodies before washing with PBS-T. The primary antibodies, including anti-NFATc1 and anti-TRAF3, were purchased from Abcam (Cambridge, UK). Finally, the membranes were detected by Odyssey Infrared Imaging System.