Amersham ecl select western blotting detection reagent

S2 cells were lysed in ice-cold IP buffer (50 mM TRIS pH 8.0, 150 mM NaCl, 1% Triton-X and cOmplete ULTRA protease inhibitor, Roche) 48 hours after transfection for total protein lysate extraction. Cell lysates were centrifuged at 18000 rpm at 4 °C to collect the supernatant and total protein concentration was estimated by Bradford assay. Blots were probed with the primary antibodies anti-Cas9 (Abcam 191468, 1:500), anti-tubulin (Abcam 18251, 1:5000), anti-FLAG (Abcam 1162, 1:4000), anti-acetyl lysine (Cell Signaling Technology # 9441, 1:1000), and HRP coupled secondary anti-mouse (P0260) and anti-rabbit (P0448) antibodies (DAKO/Agilent, both at 1:2000). Amersham ECL Select Western Blotting Detection Reagent (GE Healthcare) was used and signals imaged with a Chemi Doc XRS+ with Image Lab Software (BioRad).

NE from wild type and THOC1-3C-AID-GFP containing human K562 cells was diluted 1:1 with buffer P (40 mM HEPES pH 7.9, 15 mM KCl, 6 mM MgCl₂, 2 mM DTT). For immunoprecipitation, the NE was first incubated for 1 hr at 4°C with or without 2 µg Benzonase per mL NE, as indicated in Figure 1—figure supplement 1f, and then incubated on a rotating wheel for 2.5 hr at 4°C with GFP-Trap Agarose resin (Chromotek) that was previously equilibrated with buffer Q (20 mM HEPES pH 7.9, 100 mM KCl, 2 mM MgCl₂, 8% (w/v) glycerol, 0.05% (v/v) Igepal CA-630, 1 mM DTT). After five washes, the beads were eluted by boiling and the samples were applied to SDS–PAGE, transferred onto a PVDF membrane (ThermoScientific) and probed with anti-THOC1 primary antibody (HPA019096, Merk, dilution 1:500). Goat anti-mouse IgG-HRP (W4021, Promega, dilution 1:10000) was used as a secondary antibody. Antibody detection was performed with Amersham ECL Select Western Blotting Detection Reagent (GE Healthcare) and a ChemiDoc MP imaging system (Bio-Rad Laboratories).

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).

293T cells were transfected with equal amounts of plasmids encoding CL80 NA-wt and the stalk variants. At 16 h posttransfection, the cells were harvested with sample buffer. The samples were analyzed by SDS-PAGE and transferred onto polyvinylidene difluoride membranes (Millipore). Immunoblot analysis was performed using anti-influenza virus H9N2 NA antibody (Sino Biological) and horseradish peroxidase-conjugated secondary antibody. The bands were visualized with the Amersham ECL Select Western blotting detection reagent and an Amersham Imager 600 (GE Healthcare). The band intensities were quantified by Amersham Imager 600 Analysis software (GE Healthcare).