Anti hsp90 antibody

Proteins were extracted from cells transfected and infected as described above for the replication assays. Cells were lysed in radioimmunoprecipitation assay buffer, and the cleared lysate was run on a 12% acrylamide gel. The proteins were transferred onto a nitrocellulose membrane (GE Healthcare) and immunoblotted using anti-Rep antibody (1/100 dilution; clone 303.9; Progen), anti-Cap antibody (1/500 dilution; clone B1; American Research Products), and anti-HSP90 antibody (polyclonal, 1/5,000 dilution; Santa Cruz). All antibodies were incubated in blocking buffer (5% nonfat dried milk in PBS containing 0.1% Tween 20). Images were acquired and analyzed using an ImageQuant apparatus (GE Healthcare).

After the indicated treatment, cells were lysed in Mammalian Protein Extraction Reagent (Thermo Scientific, Rockford, IL) containing Xpert protease inhibitor cocktail (GenDEPOT, Baker, TX) and Xpert phosphatase inhibitor cocktail (GenDEPOT) and centrifuged at 12,000 rpm for 10 min at 4°C. Protein samples were separated using SDS-PAGE and subsequently transferred onto PVDF membranes. The membranes were then blocked by immersion in blocking buffer consisting of 5% bovine serum albumin in TBST (TBST is 20-mM Tris-HCl, 150-mM NaCl, 0.2% Tween 20, pH 7.4) at RT for 1 h on a shaker. The membranes were washed three times with TBST and then incubated with an anti-PPARγ antibody (Cell Signaling Technology, Danvers, MA, Cat. No. 2443), anti-adiponectin antibody (Thermo Scientific, Cat. No. MA1-054), anti-ERK antibody (Cell Signaling Technology, Cat. No. 91025S), anti-phosphoERK antibody (Cell Signaling Technology, Cat. No. 4370S), and an anti-HSP 90 antibody (Santa Cruz Biotechnology, Dallas, TX, Cat. No. SC-13119) at 4°C overnight. After washing with fresh TBST, membranes were incubated with rabbit or mouse IgG conjugated to horseradish peroxidase (Bio-Rad; 1:5,000 dilution). Protein bands were visualized using ECL reagent (Bio-Rad) and an iBright CL1500 imaging system (Thermo Scientific).

The anti-Thy-1 antibody was from BD Pharmingen Inc. (Franklin Lakes, NJ, United States) and anti-Hsp90 antibody, from Santa Cruz Biotechnology (Santa Cruz, CA, United States). The anti-β₃ Integrin polyclonal antibody was from Millipore (Burlington, MA, United States) and the secondary anti-mouse and anti-rabbit antibodies coupled to horseradish peroxidase were from Bio-Rad Laboratories (Hercules, CA, United States). Protease inhibitors were from Roche Diagnostics (Risch-Rotkreuz, Switzerland). The chemiluminescent substrate (EZ-ECL) and Protein A beads were from Pierce Chemical (Rockford, IL, United States). The siRNAs -a pool of three siRNAs targeting β₃ Integrin and a scrambled sequence used as a control- were obtained from Ambion (Austin, TX, United States). All chemicals were purchased from Sigma-Aldrich (St. Louis, MO, United States), unless otherwise indicated.

Ileum tissues were homogenized, and the resulting total protein was extracted by RIPA lysis mixed with PMSF (Solarbio, Beijing, China). Then, 50 μg of protein per sample was subjected to 7.5%, 10%, or 15% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE, Solarbio, Beijing, China). After overnight incubation at 4°C with anti-NLRP3 antibody (dilution at 1 : 1000, Abcam, Cambridge, UK), anti-caspase-1 antibody (dilution at 1 : 300, Santa Cruz, Oregon, USA, SC-398715), anti-IL-1β antibody (dilution at 1 : 600, Bioss, Beijing, China), anti-IL-18 antibody (dilution at 1 : 600, Bioss, Beijing, China), anti-β-actin antibody (dilution at 1 : 2000, Servicebio, Wuhan, China), and anti-HSP-90 antibody (dilution at 1 : 1000, Santa Cruz, Oregon, USA), the membranes with blotted proteins were then incubated with HRP-conjugated goat anti-rabbit secondary antibody (dilution at 1 : 2000, CST, Boston, USA) or rat anti-mouse secondary antibody (dilution at 1 : 2000, Servicebio, Wuhan, China) for an hour at room temperature. After washing three times with TBST, the electrochemiluminescence solution (ECL, Millipore, Massachusetts, USA) was added to the membranes, and then, the membranes were exposed to the exposure machine (ChemiScope series, Clinx Science Instruments Co., Ltd), and the resulting images were recorded and analyzed.

To compare Akt-phosphorylation levels, insulin (2 U per kg body weight) was injected via inferior vena cava under regular fed condition. 3 min after the injection, adipose tissue, liver tissue, and muscles were collected. Frozen tissue extracts were minced and homogenized in cold RIPA buffer containing 50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 1% NP40, 0.1% SDS, 1 mM EDTA, phosphatase inhibitor cocktail (Sigma-Aldrich), and protease inhibitor cocktail (Roche), and then were centrifuged at 12,000 × g for 5 min. Equal amounts of cell lysates were subjected to immunoblot analysis. Anti-Akt, anti-phospho-Akt (Ser473) antibodies were obtained from Cell Signaling. Quantification of bands on western blot was accomplished by scanning the blots, then determining the densities of the bands using ImageJ software (National Institutes of Health, Bethesda, MD, http://imagej.nih.gov/ij/, 1997–2012). Anti-Ubc13 was obtained from Zymed laboratories. Anti-HSP90 antibody was purchased from Santa Cruz Biotechnology.