Ecl western blot detection kit

Western blot analysis was performed as described previously [28 (link)]. Whole animal protein extracts were obtained from 300 gravid adult hermaphrodites of each condition per gel well. Antibodies bound to a nitrocellulose membrane (PROTRAN BA83, Whatman, Sigma-Aldrich, St. Louis, MO, USA) were visualized with an ECL Western blot detection kit (Amersham, GE Healthcare Life Sciences, Pittsburgh, PA, USA), and the band intensities were measured with the LAS-3000 image analyzer using Multi Gauge software (v.3.0, Fuji Film, Tokyo, Japan). The following primary antibodies were used: rabbit anti-CED-9, a member of the anti-apoptotic Bcl-2 gene family in C. elegans (1:1000; Santa Cruz Biotechnology, Dallas, TX, USA) and goat anti-CED-4, the pro-apoptotic Apaf-1–like cell-death activator (1:1000; Santa Cruz Biotechnology, Dallas, TX, USA). The mouse anti-α-tubulin was used as the loading control (1:1000; Sigma-Aldrich, St. Louis, MO, USA). The following secondary antibodies were used: horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG (1:1000; Santa Cruz Biotechnology, Dallas, TX, USA), HRP-conjugated donkey anti-goat IgG (1:1000; Santa Cruz Biotechnology, Dallas, TX, USA), and HRP-conjugated donkey anti-mouse IgG (1:1000; Jackson ImmunoResearch, West Grove, PA, USA).

Proteins were subjected to Coomassie blue quantification before use in western blotting with some modification of published methods (Ibe et al. 2000, 2002, 2007). Proteins were suspended in sodium dodecyl sulfate (SDS) sample buffer, pH 6.8, containing: 125 mmol/L Tris‐base, 4% SDS, 0.006% bromophenol blue, 36 mmol/L EDTA, 90 mmol/L DTT, 10% glycerol, 10% beta‐mercaptoethanol. Proteins were loaded on each sample per lane and subjected to SDS‐PAGE gel electrophoresis for 1 h at 200V on 4–12% Tris‐glycine gradient gels (Lonza, Rockland, ME), along with Bio‐Rad kaleidoscope prestained molecular weight markers and protein standards. After 1 h of SDS‐PAGE, proteins were transferred to nitrocellulose membrane by means of mini Trans‐Blot, (Bio‐Rad, Irvine, CA) at 70V and then blocked with 5% nonfat dry milk in 1% T‐TBS overnight. Blots were then incubated for 2–4 with the specific antibody, for instance, 1:500 of PAFR antibody; then, each antibody was washed with 1% T‐TBS, incubated for 1 h with an anti‐rabbit Ig HRP‐linked secondary antibody (Amersham), followed by three more washes with 1% T‐TBS. The signals were developed for 1 min using Amersham ECL Western Blot detection kit and then exposed to X‐ray film. Bands corresponding to PAFR and PKA‐Cα proteins were scanned and quantified.

Tat protein expression from the recombinant vector was analysed in both BALB/c and Vero cells (1×10⁶ cells) infected with HSV1-Tat at 1 MOI. Negative controls were uninfected cells, and cells infected with 1 MOI of the HSV1-LacZ control vector. Cell extracts, corresponding to 10 µg of total proteins, were loaded into 12% SDS-polyacrylamide gels and analysed by Western blot using a rabbit anti-Tat polyclonal serum (Intracel, Cupertino, CA) at 1∶1,000 dilution and mouse anti-rabbit horseradish peroxidase (HRP)-conjugated secondary antibody (Sigma-Aldrich) at 1∶4,000 dilution. Immunocomplexes were detected by means of the ECL Western Blot detection kit (Amersham Pharmacia). A recombinant Tat protein (1 µg), obtained from Diatheva (Urbino, Italy), was included in each gel as positive control.

Lysates from transfected or infected cells were prepared in a sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) loading buffer, separated on sodium dodecyl sulfate-containing 8 to 15% polyacrylamide gels, and transferred to nitrocellulose membranes. Chemiluminescence was detected according to the manufacturer's protocol (ECL Western blot detection kit; Amersham Pharmacia Biotech).

Individual hippocampi were homogenized in RIPA buffer (50 mM Tris-HCl, 150 mM NaCl, 1% NP-40, 0.5% sodium deoxycholate, and 0.1% SDS, pH 7.4) supplemented with protease inhibitor cocktail tablets (Roche). Homogenates were centrifuged at 14,000 × g for 15 min and the supernatants were used for immunoblotting. Protein concentrations were determined using BCA Protein Assay Kit (Thermo Scientific, Rockford, IL). Samples were separated on 4–15% SDS-PAGE and transferred onto PVDF membranes (Bio-Rad Laboratories, Hercules, CA). Membranes were blocked for 1 h at room temperature in 3% (w/v) bovine serum albumin (BSA) in TBST buffer (50 mM Tris-Cl, pH 7.6; 150 mM NaCl; 0.1% (v/v) Tween-20), and incubated overnight at 4 °C with the respective antibodies as follows: rabbit anti-ZO-1 (1:1000, Invitrogen), mouse anti-occludin (1:1000, Invitrogen), rabbit anti-DCX (1:1000, Abcam), mouse anti-NeuN (1:1000, Millipore), mouse anti-fibrinogen β (1:1000, Santa Cruz Biotechnology) and monoclonal anti-β-actin-peroxidase (1:50000; Sigma-Aldrich). Individual immunoblots were visualized by an ECL Western blot detection kit (Amersham Biosciences, Piscataway, NJ), the proteins of interest were quantified by ImageJ software (National Institutes of Health, Bethesda, MD), normalized to actin expression, and used for statistical analysis.