Rabbit monoclonal anti flag

After PBS washings, transfected HeLa cells grown on chamber slides were fixed with 4% PFA for 15 min. After PBS washing cells were incubated in 50/50 acetone/methanol for 20 min. Mouse monoclonal anti-V5 antibody (Life Technologies), and rabbit monoclonal anti-FLAG (Sigma–Aldrich) were incubated at 1/200 for 1 h at room temperature, followed by incubation with a mouse specific Alexa-488 goat antibody and rabbit specific alexa-555 conjugated donkey antibody 1 h at room temperature in the dark. After washing, slides were mounted with Vectashield imaging medium containing DAPI (Vector Laboratories). Imaging was performed using Leica SP5 confocal microscope.

SV589 cells were set up on glass coverslips at 1.5 × 10⁵ cells per six-well plate in 2 ml medium C with 5% FCS. At 24 h after plating, cells were transfected with 1 µg of the indicated plasmid using FuGENE HD as the transfection agent. At 24 h after transfection, cells were fixed for 15 min in 3.7% formaldehyde in PBS at room temperature and permeabilized for 10 min in methanol at −20 °C. After blocking by incubation with 1 mg/ml bovine serum albumin in PBS, cells were double-labeled with mouse monoclonal anti-LAMP-2 (1: 100, BD biosciences, 555803) and rabbit monoclonal anti-Flag (1: 100, Sigma-Aldrich, F7425) followed by goat anti-rabbit IgG conjugated with AlexaFluor 488 (1: 300, Ivitrogen, A-11008) and goat anti-mouse IgG conjugated with AlexaFluor 594 (1: 300, Invitrogen, A-11005). The coverslips were then mounted in Shandon Immu-mount (Thermo Scientific) and fluorescence images were acquired using a Plan-Apochromat ×63/1.4 oil DIC objective (Zeiss, Oberkochen, Germany), a Zeiss LSM 800 microscope (Zeiss), and ZEN imaging software (Zeiss).

Adherent cells were mechanically scraped in PBS in the presence of 5 mM EDTA and resuspended in lysis buffer (0.5% Igepal, 0.5% Triton X-100 and 5 mM EDTA in PBS) supplemented with protease inhibitors cocktail (PMSF, benzamidine, aprotinin, and leupeptin). Quantification of total proteins was performed with Bradford reagent (BIO-RAD, Kidlington, UK), using bovine serum albumin as standard. For the SDS-PAGE analysis, 70 μg of intracellular lysate were loaded. Immunoblot analysis was performed using standard procedures and detected with a chemiluminescent substrate, WESTAR ETA C ULTRA 2.0, Cyanagen (Sigma Aldrich, St. Louis, MO, USA). Antibodies used were: Rabbit monoclonal anti-FLAG (Sigma Aldrich); HRP anti-rabbit IgG (Jackson ImmunoResearch Laboratories, West Grove, PA, USA).