Ab6829

Antibodies (Ab)s for flow cytometry were purchased from Becton Dickinson ((BD), NJ, USA): anti-CD 61 (integrin beta 3 chain), PerCP (clone RUU-PL 7F12 hybridization of mouse P3X63.Ag8.653 myeloma cells with spleen cells from BALB/c mice immunized with purified platelet membrane glycoproteins) (CD61 PerCP (340506)), and a fluorescein isothiocyanate- (FITC-) conjugated monoclonal antibody to CD41a (clone HIP8, derived from hybridization of mouse P3X63 myeloma cells with spleen cells from BALB/c mice immunized with purified platelet membrane glycoprotein) (CD41a FITC (340929)). All Abs for western blotting were purchased from Abcam (England, UK): (primary antibodies (1°): anti-P2Y₁₂ Ab (rabbit polyclonal to P2Y₁₂) (ab82725), dilution 1 : 1000; anti-beta actin (rabbit polyclonal to beta-actin-loading control) (ab8227), dilution 1 : 2000; and secondary antibody (2°): chicken polyclonal secondary Ab to rabbit IgG-H&L (HRP) (ab6829), dilution 1 : 2000). The Aurum serum protein mini kit and protein standards and ladder for SDS-PAGE (Precision Plus Protein Western Standards Value Pack) were purchased from Biorad (USA). The enhanced chemiluminescence detection kit (Amersham ECL Select western blotting detection reagent) was purchased from GE Healthcare life sciences (UK).

Samples were directly lysed in Laemmli Buffer and separated by SDS-PAGE on a 12% polyacrylamide gel. Separated proteins were transferred to Hybond nitrocellulose membranes (Amersham Biosciences, Amersham, UK) and blocked in 5% milk in Tris-buffered saline (milk-TBS). Primary antibodies were diluted in milk-TBS as follows: cleaved caspase-3 (CST, #9664, 1/500), PARP (CST, #9542, 1/1000), β-actin (Abcam, ab8227, 1/2000), SOD2 (Abcam, ab13533, 1/1000), and GPX-1 (Abcam, ab22604, 1/1000), and incubated with membranes overnight at 4 °C. Secondary antibody (chicken anti-rabbit-HRP, Abcam, ab6829, 1/2000) was incubated with membrane for 1 hr at room temperature. Blots were incubated with Amersham ECL Western Blotting detection reagent (GE Healthcare Life Sciences) for 5 min and then exposed to autoradiography film (Fujifilm, Tokyo, Japan). All densitometry analysis was performed using ImageJ software.