Anti rabbit igg secondary antibody

Samples of Triton X-100-lysed ghost membranes (30 µg) were run (90 min at 180 V, room temperature) in a SDS-polyacrylamide gel (10%, 1 mm thickness) under reducing conditions (570 mM βME). Running buffer: 250 mM Tris, 192 mM glycine, 5% SDS, pH = 8.3. Transfer to nitrocellulose membranes (90 min at 30 V, room temperature) was performed in transfer buffer containing 12.5 mM Tris, 96 mM glycine, 20% ethanol, 0.1% SDS, pH = 8.5. Immunoblotting was performed using rabbit anti-hVDAC1 antibody (Santa Cruz Biotechnology, B-6, dilution 1:1000), or rabbit anti-ANT polyclonal antibody (a generous gift from Dr G. Brandolin, CNRS, CEA, Grenoble, France, dilution 1:1000^{64 (link)}). Western blotting secondary labelling was performed with an anti-rabbit IgG secondary antibody (Jackson ImmunoResearch, dilution 1:5000). Loading control was performed using HRP Conjugated anti-β-Actin mAb (Cell Signaling Technology, 13E5, dilution 1:3000).

Antibody against p-CDK1 (Thr 161) was purchased from Abcam (Cambridge, MA, USA). Anti-p85α, anti-Akt, and p-Akt (Ser 473) antibodies were purchased from BD PharMingen. Antibodies against p21, CDK1, CDK2, cyclin A, cyclin B1, cyclin D, cyclin E, and p110α were purchased from Santa Cruz Biotechnology. Anti-pan-cadherin, anti-PTEN, and anti-PTEN (Ser 380/Thr 382/385) antibodies were obtained from Thermo Fisher Scientific (New York, NY, USA). Antibodies against β-actin, hemagglutinin (HA)-epitope tag, and FLAG-epitope tag were obtained from Sigma-Aldrich. Peroxidase-conjugated anti-mouse IgG, anti-goat IgG, and anti-rabbit IgG secondary antibodies were purchased from Jackson ImmunoResearch Laboratory (West Grove, PA, USA).

Biopsies were cut into 1-mm³ blocks and fixed in 2% formaldehyde and 0.05% glutaraldehyde for 1 h at room temperature. Samples were dehydrated in 30% ethanol at 0 °C, 55% ethanol at −15 °C, 70% ethanol at −30 °C and 100% ethanol at −50 °C and gradually infiltrated in 100% Lowicryl HM20 (Polysciences, Germany). Samples were polymerized under UV light. Ultrathin sections were cut and collected on gold grids. Nonspecific labelling was blocked with PBS buffer containing 0.1% fish gelatine, 0.8% BSA and 5% FCS. Then, UPs were immunolocalized by anti-UPs primary antibody (1:5000). After washing with PBS, UPs were detected with anti-rabbit IgG secondary antibodies conjugated to 5 nm or 10 nm gold (Jackson ImmunoResearch, Cambridgeshire, United Kingdom). Sections were washed with PBS followed by distilled water. Five nm gold was silver-enhanced for 4 min with IntenSE (Amersham, UK). Ultrathin sections were counterstained with uranyl acetate and lead citrate (Merck, Darmstadt, Germany). For negative controls the protocols were the same, except omitting the primary antibody or incubating the sections with rabbit serum. Sections were analysed with transmission electron microscope (Philips CM100, The Netherlands) at 80 kV and micrographs were taken with an AMT camera (Advanced Microscopy Techniques Corp., Woburn, MA, USA).

Anti-ING4 (C-term) polyclonal antibody was purchased from Abgent. Mouse monoclonal antibodies against histone H3, trimethylated histone H3 lysine 4 (H3K4me3) and acetylated histone H3 lysine 9 (H3K9ac) were from Takara Bio. Monoclonal anti-nucleolin and -UBF antibodies were from Santa Cruz. Anti-green fluorescent protein (GFP) polyclonal rabbit antibody was from MBL Life Science. Horseradish peroxidase-conjugated anti-mouse IgG and anti-rabbit IgG secondary antibodies were from Jackson ImmunoResearch. Chemicals were purchased from either Sigma or Wako, unless otherwise stated. Chronic myelogenous leukemia HAP1 cells (Horizon) were grown in Iscove’s Modified Dulbecco’s Medium (IMDM). Osteosarcoma U-2 OS and cervix adenocarcinoma HeLa S3 cells (both from ATTC) were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM). All media were purchased from Nacalai Tesque. All the cells were cultured in the medium supplemented with 10% fetal bovine serum (FBS) (Gibco), 100 unit/ml penicillin and 100 μg/ml streptomycin (Nacalai Tesque) at 37 °C under an atmosphere of 5% CO₂.