Fig. 5
From: Melanoma Differentiation Associated Gene-7 (mda-7): A Novel Anti-Tumor Gene for Cancer Gene Therapy
Confocal analyses of MDA-7 subcellular localization and secretion of soluble MDA-7. (A) Confocal studies. (I) shows surface labeling of MDA-7 protein in H460 cells (red), 48 hours post-Ad-mda7 or Ad-luc infection (MOI of 1000 vp/cell). (II) shows the same H460 cells stained with Annexin V-FITC (green). (B) H460 cells were transduced with 1000 (I) or 5000 (iI) MOI of Ad-luc or Ad-mda7, and 48 hr later, cells were incubated with anti-MDA7 rabbit polyclonal antibody followed by goat anti-rabbit IgG-tagged FITC (Fluoroscein Iso-thiocynate tag). (C) HeLa cells were infected with 500 vp/cell of Ad-mda7. The cells were fixed with 2% para-formaldehyde and treated with 0.001% Triton X-100 for permeabilization. (I) represents the expression of MDA-7 protein (green fluorescence; using anti-MDA-7 rabbit polyclonal as the primary and anti-rabbit IgG-FITC as the secondary antibody) as a distinct punctate, cytoplasmic staining. (II) represents the same field showing actin fibres stained with Phalloidin Alexa 594. (III) represents the composite of (I) and (II). (D) Western blot analysis of Ad-mda7 transduced H1299 cell lysate (lane 1) and cell supernatant (lane 3); Ad-luc transduced cell lysate (lane 2) and cell supernatant (lane 4) were used as controls. The cells were infected with 1000 vp/cell of Ad-mda7 or Ad-luc, and 48 hr later, the cell lysate and 0.22 um filtered supernatant were analyzed using the rabbit anti-MDA-7 polyclonal affinity purified antibody. (Figure continued on next page)