CXCL4 and CXCL4L1 expression by HEK-293 and HMVEC transfectants. (A) CXCL4 and CXCL4L1 mRNA expression by HEK-293 transfectants, as assessed by real-time qRT-PCR. Columns represent mean values (± SEM) of 10 selected clones. (B) Expression of CXCL4/CXCL4L1 in HEK-293 transfectants, as detected by immunofluorescence. Absence of signal in mock transfectants (left), strong cytoplasmic signal in CXCL4 transfectants (green, middle panel), staining in a region adjacent to plasmatic membrane in CXCL4L1 transfectants (green, right). Bar represents 50 μm. (C) High-power magnification of immunofluorescence performed on CXCL4 (left) or CXCL4L1 transfectants (right). Bar represents 20 μm. (D) Confocal microscopy of HMVECs transfected with CXCL4-GFP. GFP fluorescence (green) was localized in the cytoplasm; concanavalin A stains the membrane (red). Merged image (yellow) demonstrates absence of coexpression; bar represents 20 μm. At the extreme part of the figure a high-power magnification of a detail of the merged image is shown; bar represents 5 μm. (E) Confocal microscopy of HMVECs transfected with CXCL4L1-GFP. GFP fluorescence (green) was localized in the cytoplasm and on cytoplasmic membrane, as demonstrated by merged image (yellow; bar represents 20 μm). At the extreme part of the figure a high-power magnification of a detail of the merged image is shown (bar represents 5 μm). Images in panels B and C were acquired using an LSM 510 Meta confocal microscope (Zeiss) equipped with a 20 ×/0.50 NA Plan-Neofluor objective lens (Zeiss). Images in panel D and E were acquired using an LSM 510 Meta laser scanning confocal microscope equipped with a 40×/1.30 NA oil Plan-Neofluor objective lens. An electronic zoom is shown in the extreme panel. LSM 510 Meta confocal microscope software version 3.0 (Zeiss) was used to capture all the images.