Enhanced chemiluminescence ecl reagent

RAW264.7 cells and HEK293T cells were purchased from the American Type Culture Collection (ATCC) (Rockville, MD, USA). Roswell Park Memorial Institute (RPMI) 1640, Dulbecco’s modified Eagle’s medium (DMEM), and penicillin–streptomycin solution were purchased from Hyclone (Logan, UT, USA). Fetal bovine serum (FBS), Opti-MEM reduced serum medium and TRIzol were purchased from GIBCO (Grand Island, NY, USA).
LPS, L-NAME, ranitidine, Pam3CSK4, Poly(I:C), dimethyl sulfoxide (DMSO), (3-4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT), polyethylenimine (PEI), sodium dodecyl sulfate (SDS), and O-nitrophenyl-beta-D-galactopyranoside (ONPG) were acquired from Sigma Chemical Co. (St. Louis, MO, USA). TRI reagent^® was acquired from Molecular Research Centre Incorporated (Cincinnati, OH, USA). Enhanced chemiluminescence (ECL) reagent was supplied by Amersham (Bath, UK). PCR primers specific for COX-2, iNOS, IL-6, TNF-α, and GAPDH were obtained from Macrogen (Seoul, Korea). Antibodies specific for phosphorylated and total forms of p65, p50, IκBα, p85, AKT, Src, Syk. human influenza haemagglutinin (HA), and β-actin were purchased from Cell Signaling Technology (Beverly, MA, USA).

LPS, dexamethasone, Bay 11-7082, and p-nitrophenyl phosphate (pNPP) were purchased from Sigma (St. Louis, MO); all chemicals were >98% pure. Various culture media and supplements were obtained from Invitrogen Technologies (Carlsbad, CA). Fetal calf serum was from Hyclone (Thermo Fisher Scientific, Waltham, MA). Antibodies against iNOS and COX-2 were purchased from Abcam (Cambridge, UK) and Cayman (Ann Arbor, MI) respectively. The antibody against glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was obtained from Abcam Ltd. The enhanced chemiluminescence (ECL) reagent was purchased from Amersham Biosciences (Piscataway, NJ). Penicillin, streptomycin, and horseradish peroxidase (HRP)-conjugated anti-mouse secondary antibodies were purchased from Invitrogen Technologies.

Whole brain tissues were homogenized in protein extraction solution (Intron, Seoul, Korea) containing 1% protease inhibitor cocktail (Thermo Fisher Scientific, Rockford, IL, USA) using a tissue blender and centrifuged at 12,000 rpm for 20 min at 4 °C. To equalize total protein amounts, protein concentrations in supernatants were measured using the Bradford protein assay (BioRad Laboratories, Hercules, CA, USA). Proteins (30 μg) separated using SDS gels and then transferred to polyvinylidene difluoride (PVDF) membranes (Millipore, Billerica, MA, USA). After blocking with 5% skim milk in tris buffered saline containing Tween-20 (TBST), membranes were incubated overnight with the following primary antibodies at 4 °C; actin, p-IRS-1 (Tyr 612), IRS-1, PI3K, p-Akt (Ser 473), Akt, AChE, GLUT4, Bax, Bcl-2, p-Tau (Ser 404), and Tau (all dilutions 1:1000). Membranes were then washed with TBST 5 min and incubated with horseradish peroxide (HPR)-conjugated secondary anti-mouse or anti-rabbit antibodies (1:3000) in the presence of 5% skim milk in TBST for 1 h at room temperature. For chemiluminescence detection, the membranes were visualized using enhanced chemiluminescence ECL reagent (Amersham Pharmacia, Piscataway, NJ, USA). Band densities were calculated using image making software (ImageJ; National Institute of Health, Bethesda, MD, USA).

AF, N-acetyl-L-cysteine (NAC), Tertiary butylhydroquinone (Tbhq), glutathione ethylene ester (GEE), ascorbic acid (Vitamin C), Annexin V, propidium iodide (PI) and rhodamine-123 were obtained from Sigma-Aldrich (St. Louis, MO). DCF-DA and z-VAD-fmk were from BD Biosciences (San Jose, CA). Antibodies (Abs) against c-Abl (C-19), ubiquitin (P4D1), Mcl-1 (S-19), caspase-3, -8, -9, apoptosis-inducing factor (AIF), Bcl-2, Bax, GAPDH (FL-335) were from Santa Cruz Biotechnology (Santa Cruz, CA). Antibodies against poly (ADP)-ribose polymerase (PARP, clone 4C10-5) was from BD Biosciences. Antibodies against phospho-c-Abl at Y245, phospho-Erk1/2 (T202/Y204), Erk1/2, phospho-Akt, Akt, IκB-α, Bcl-xL, survivin, cleaved caspase-3, -9, cytochrome C, and XIAP were from Cell Signaling Technology (Beverly, MA, USA). Antibodies against phospho-STAT5A/B (Y694/Y699, clone 8-5-2), STAT5, and Ki67 were from Upstate Technology. Enhanced chemiluminescence (ECL) reagents were purchased from Amersham Biosciences (Piscataway, NJ, USA).

Generally 1.5 × 106 EAHY cells were inoculated onto 10-cm culture dishes and exposed to various concentrations of 7-KC or DMSO for 24h. Cell lysates were prepared by dissolving cells in lysis buffer (10 mM Tris-HCl, pH 7; 140 mM sodium chloride; 3 mM magnesium chloride; 0.5% NP-40; 2 mM phenylmethylsulfonyl fluoride; 1% aprotinin; and 5 mM dithiothreitol) and the same amounts of proteins (20-50 μg/ml) were loaded to 12.5% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) for protein separation and transferred to a polyvinylidene fluoride (PVDF) membrane. The membrane was blotted first with primary antibodies against Cdk1, cyclin B1 and GAPDH for 2 hr as described previously [36 (link), 37 (link)]. This was followed by incubation with respective horseradish peroxidase-link secondary antibodies (Jackson ImmunoResearch Laboratories, West Grove, PA, USA) for 1 hr. After rinsed the membrane with buffer, Enhanced chemiluminescence (ECL) reagents (Amersham, Piscataway, NJ, USA) were added and the chemiluminescence was detected by exposure of membranes to Fuji films for 30 sec to 10 min. The intensity of GAPDH bands was used as control.

Dulbecco’s modified Eagle’s medium, Minimal Essential Medium Eagle alpha modification (α-MEM), neomycin, fetal bovine serum, horse serum, and penicillin-streptomycin were obtained from Thermo Fischer Scientific (Waltham, MA, USA). Collagenase type II was from Worthington Biochemical (Lakewood, NJ, USA). Complete™ Mini protease inhibitors were from Roche Applied Science (Indianapolis, IN, USA). Protein A/G agarose was from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Mouse anti-Strep antibody was from IBA GmbH (Gottingen, Germany), anti-caveolin-3 was from BD Bioscience (San José, CA, USA), and anti-Dys was from Leica Biosystems (Wetzlar, Germany). Rabbit anti-P2Y₂R and anti-Panx1 antibodies, mouse anti-DHPR α-1 antibody, and secondary anti-mouse and anti-rabbit antibodies conjugated to either horseradish peroxidase or Alexa Fluor® dyes were from Thermo Fischer Scientific. Enhanced chemiluminescence (ECL) reagents were from Amersham Biosciences (Piscataway, NJ, USA). ECM (gel from Engelbreth-Holm-Swarm murine sarcoma) matrix was from Sigma-Aldrich (St. Louis, MO, USA). All other reagents used were of analytical quality.

Rabbit anti-HDAC1 was purchased from Diagenode (Belgium), rabbit anti-H3 and rabbit anti-H3Ac were obtained from Abcam (Cambridge, UK). All primary antibodies were used according to the manufacturer's instructions. Horseradish peroxidase-conjugated secondary antibodies were from Sigma-Aldrich and enhanced chemiluminescence (ECL) reagents were from Amersham Biosciences (Piscataway, USA). All other reagents were purchased from Sigma-Aldrich, unless otherwise stated.