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. 2008 Jan 16;82(6):2715–2726. doi: 10.1128/JVI.02456-07

FIG. 3.

FIG. 3.

(A) Partial colocalization of mEGFP-huPSS-1 and dsRed-1-Mito markers. PSS-120 cells expressing mEGFP-huPSS-1 were transiently transfected with a plasmid expression vector for DsRed-1-Mito. After 24 h they were fixed and probed with mouse anti-GFP (1:200). The secondary antibody was FITC-goat anti-mouse (1:250, green). The cells were imaged for emissions at both 520 nm (FITC, left) and 615 nm (TR, middle) by using confocal microscopy. The overlaid image is on the right. (B) Diffuse mEGFP localization in transfected HeLa cells. HeLa cells were transfected with plasmids expressing mEGFP (pmEGFP-C1) and DsRed-1-Mito. Cells were fixed 24 h later and examined as in panel A for the presence of mEGFP and DsRed-1- mito. Fixed cells were examined for DsRed (top) or probed with human autoimmune anti-mitochondria antibodies (1:50) (bottom). Secondary antibody was TR-goat anti-human IgG (1:50). Confocal images were taken sequentially through a ×100 objective lens. Zeiss Lasersharp 2000 Acquisition software was used for additional magnifications. (C) Colocalization of pUL37x1 with mEGFP-huPSS-1 and with dsRed-1-Mito. PSS-120 cells expressing mEGFP-huPSS-1 were transiently transfected with DsRed-1-Mito expression vector and a pUL37x1 expression plasmid (p327). After 24 h they were fixed and probed with mouse anti-GFP (1:200) and rabbit anti-UL37x1 (Ab1064, 1:250). The secondary antibodies were FITC-goat anti-mouse (1:250, green) and Cy5-Goat anti-rabbit (1:50, blue). Cells were imaged by using confocal microscopy at 520 nm (FITC, top row, left panel), 615 nm (TR, top row, middle panel), and 670 nm (Cy5, top row, right panel). Panels also show the overlaid images of green-red (center row, left), green-cyan (center row, middle), red-cyan (center row, right) and green-red-cyan (bottom row, left). The bottom right panel shows detail of the boxed area from the triple overlay. (D) Lack of colocalization of mEGFP and pUL37x1 in transfected HeLa cells. HeLa cells were transfected with expression plasmid for mEGFP (pmEGFP-C1) and for pUL37x1 (p327). Cells were fixed 24 h later and examined as panel C for the presence of EGFP and for pUL37x1 by using Ab1064 (1:300) and TR-goat anti-rabbit IgG (1:300). Cells were imaged by using confocal microscopy at 520 nm (FITC, left panel) and 615 nm (TR, middle panel). The overlaid image is on the right.