Mouse monoclonal anti ha

MCF-7 and MCF12F cells untreated, or transfected with CPT1Av1- or CPT1Av2- pcDNA-ha or alternatively with siRNA (120 nM) as reported above, were plated in 4 wells/chamberslides at a concentration of 25,000 cells/cm². After an over-night culture, were fixed in formalin 10%. The primary antibodies used were anti-CPT1Av1 (H95-Santa Cruz), mouse monoclonal anti-Ha (Covance) and anti-acetyl histone H4 (Upstate). To assess the background staining, a negative control was carried out without addition of primary antibody. Secondary antibody (biotinylated goat anti-rabbit IgG) and following reagents (HRP-conjugated streptoavidin) were added. After washing, slides were incubated with diaminobenzidine (DAB) and counterstained with haematoxylin.

Cells were fixed with 4% paraformaldehyde. Immunofluorescence assays were performed and analysed by confocal microscopy using the antibodies described below. The slides were examined with a Leica TCS SP8 or Zeiss LSM870 confocal fluorescent microscope. Protein extracts were prepared by lysing cells directly in Laemmli buffer. SUMO conjugates and PML proteins were separated on 4–12% gradient SDS–PAGE (Biorad). Homemade chicken polyclonal anti-human PML was previously described^{41 (link)}. Mouse monoclonal anti-HA was from Covance, anti-GFP from Roche, rabbit polyclonal anti-CFP and goat polyclonal anti-lamin B antibodies from Sigma-Aldrich. Anti-mouse cKit (CD117) and anti-mouse Mac1 antibodies were from BD pharmingen. Alexa 488- or 594-labeled secondary antibodies and HRP-conjugated secondary antibodies from Jackson Laboratories.
For statistics, PML NBs were counted from at least 50 randomly chosen cells. The ratio between sumoylated and unmodified PML was calculated from Vilber Lourmat Fusion camera software. All experiments have been done at least with three independent replicates.

To quantify cell surface expression of Kir2.1 in mammalian cells, 36 h after transfection COS7 were fixed with 3% paraformaldehyde (PFA) for 15 min on ice, blocked on ice with blocking buffer (5% Fetal Bovine Serum in 1× PBS, 30 min), incubated with mouse monoclonal anti-HA (Covance Inc.; 1:300 dilution in blocking buffer, 1 h at room temperature), washed (with 1× PBS, three times for 5 min), incubated with goat anti-mouse IgG HRP-conjugated secondary antibody (1:1000 dilution, Jackson) (in blocking buffer, 30 min), and then extensively washed (1× PBS, 5 min/time for four times in total). After washing, the cells were scraped off from the plates and resuspended well into 500 μl 1× PBS. From the cell suspension, 10 μl of cell suspension was withdrawn and incubated with 100 μl of mixed SuperSignal ELISA Pico solution (Pierce Biotechnology, Inc., Rockford, IL, United States), and subsequently chemiluminescence signal was measured as previously described (Ma et al., 2007 (link)). To normalize the data for total protein expression level, western-blot analyses were carried out in parallel with anti-HA, or anti-Kir2.1 (Chemicon Inc.), and then quantified by densitometry analysis. Reported values are the average of triplicate transfections from three different experiments.