β actin antibody

Following protein isolation, equivalent amounts of protein were loaded onto a 10% (for inducible nitric oxide synthase (iNOS)) and 12% (for caspase-3) polyacrylamide gel for electrophoresis and blotting. After being blocked for 1 h at ambient temperature, the membrane was incubated overnight at 4°C with polyclonal anti-mouse iNOS antibody (BD Transduction Laboratories, Lexington, KY, USA) or polyclonal anti-rabbit cleaved caspase 3 antibody (Cell Signaling Technology, Danvers, CO, USA) at a 1: 1000 dilution. The blots were washed and incubated with horseradish-peroxidase-coupled secondary antibody (diluted 1∶1500; BD Transduction Laboratories, Lexington, KY, USA) for 1 h at ambient temperature. The blots were then stripped and reprobed with β-actin antibody (1∶3000; BD Transduction Laboratories, Lexington, KY, USA) as internal control. Immunoreactivity was visualized using an enhanced chemiluminescence reaction kit (Amersham Pharmacia Biotech, Buckinghamshire, UK) and quantified by scanning densitometer.

Apigenin (purity≧99%) was purchased from Extrasynthese (Genay, France); dimethylsulfoxide (DMSO), sodium dodecyl sulphate (SDS), phenylmethylsulfonyl fluoride, and bovine serum albumin (BSA) were purchased from Sigma-Aldrich Chemical Co. (St. Louis, MO, USA); the Annexin V-Alexa Fluor 488 and propidium iodide (PI) apoptosis detection kit were purchased from Invitrogen (Molecular Probe, Inc, Eugene, OR, USA). The protein assay kit was obtained from Bio-Rad Labs. (Hercules, CA, USA). Dulbecco’s phosphate-buffer saline (PBS), and trypsin-EDTA were purchased from Gibco-BRL (Gaithersburg, MD, USA). Mouse- or rabbit-monoclonal antibodies specific for cytochrome c, caspase-3, caspase-7, caspase-9, Cyclin B1, Cyclin E, and CDK2 were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Mouse- or rabbit-monoclonal antibodies specific for phospho-p53, p53, p21, p27, Bcl-2, Bcl-xL, Mcl-1, Bax, Bad, Bak, poly( ADP-ribose) polymerase (PARP), Cdc2, Cdc25C, and Cyclin A were purchased from Invitrogen Corporation (Camarillo, CA, USA). β-Actin antibody was purchased from BD Transduction Laboratories (San Diego, CA, USA). The enhanced chemiluminescence (ECL) kit was purchased from Amersham Life Science (Amersham, UK).

To analyse the expression of NLRP3 inflammasome, primary PMs were incubated for 3 h after seeding in the culture plate. Macrophages were assayed in opti‐MEM media (Gibco, 31985) in the presence or the absence of trehalose or saccharin 15 min prior to LPS stimulation (Invivogen, tlrl‐eklps; 10 ng/ml) for 3 h followed by adding ATP (Sigma–Aldrich, A6419; 5 mM) for 30 min to release IL‐1β. To confirm Atg7 protein deficiency in macrophage of Atg7^fl/fl or Atg7^fl/fl;Lyz2‐Cre mice, PMs were harvested, seeded, incubated for 3 h, and the adherent macrophages were collected for further analysis. Human T1R3 was analysed from cell lysates obtained from THP‐1 cell line or PBMCs, as described.⁸⁶ Proteins in the culture supernatant were concentrated by chloroform‐methanol precipitation, as described previously.⁹⁰ Primary antibodies used were goat anti‐mouse IL‐1β antibody (R&D Systems, AF‐401‐NA; 1:1000), mouse monoclonal anti‐NLRP3 antibody (Adipogen, AG‐20B‐0014; 1:1000), mouse monoclonal anti‐caspase‐1 antibody (AdipoGen, AG‐20B‐0042; 1:1000), rabbit anti‐Atg7 antibody (Cell Signaling, 2631; 1:1000), rabbit anti‐human T1R3 (abcam, ab229015; 1:1000), and mouse anti‐β‐Actin antibody (BD Biosciences, 612657; 1:5000).