Stable expression of FLAG-tagged ADTRP in ECs. (A) Subpanel i: validation of the anti-FLAG mAb by Western blot of HEK293T lysates, expressing or not ADTRP-FLAG. Lane 1: positive control; lane 2: negative control (both purchased from OriGene). Subpanel ii: Total lysates of EAhy926 cells were analyzed by SDS-PAGE and immunoblotting with rabbit anti-ADTRP IgG and mAbs anti-FLAG and anti–ß-actin. Lane 1: mock-transfected ECs; lane 2: EC line stable expressing ADTRP-FLAG. (B) Cell surface immunostaining with either anti-ADTRP or anti-TFPI (Cy3, red for both) and anti-FLAG (Cy5, blue) IgGs on HUVEC line stable expressing ADTRP-FLAG. Overlap (white) designates the colocalization channels resulting from the image analysis. (C) Scatter-plot and statistics (1-way ANOVA) of MFI after immunostaining for cell surface ADTRP and TFPI on control (mock-transfected) and ADTRP-FLAG–expressing HUVECs. Data are mean ± SEM. (D) Immunofluorescence on nonpermeabilized EAhy926 cell line stable expressing ADTRP-FLAG shows the cell surface distribution of TFPI (Cy3, red), ADTRP (FITC, green) and FLAG (Cy5, blue) and their double and triple colocalization channels. Bars: 10 μm. (E) Flow cytometric analysis of FLAG and TFPI on the cell surface of control cells and ADTRP-FLAG ECs. The fluorescent profile of mAb anti-FLAG is indistinguishable from the isotype-matched IgG (shaded area) on control cells (red), but shifts on ADTRP-FLAG ECs (blue, top panel). The bottom panel shows the shift in anti-TFPI staining in ADTRP-FLAG ECs (blue) compared with control cells (red).
