Endogenous Slp4-a, Rab3B, and Rab3D are expressed in HUVECs and localize to WPBs. (A) HUVECs immunolabeled for endogenous VWF (red) and Slp4-a (green). The intensity levels of the red (VWF) channel were adjusted from 0-255 to 0-130 intensity units. Scale bar represents 200 μm. Grayscale images are from the region indicated by the white box (scale bar represents 20 μm). (B) Left: HUVECs immunolabeled for endogenous Rab27A (red), Slp4-a (green), and MyRIP (blue). Scale bar represents 20 μm. Dashed white lines indicate approximate cell boundaries and a cell with few (*) and a cell with many (**) WPBs are indicated. Right top: Grayscale images are shown for each channel as indicated and the subregions i and ii shown on expanded scales below. (C) HUVECs immunolabeled for endogenous Rab3B (mouse Ab, green) or Rab3D (mouse Ab, green) and VWF (rabbit Ab, red). Top: Endogenous Rab3B-IR is localized to WPBs. Middle: > 95% of cells had no WPB-Rab3D-IR (see also supplemental Figure 4Civ). Bottom: < 5% of cells had weak WPB-Rab3D-IR (arrowheads). Scale bars represent 10 μm. Immunofluorescence images of fixed cells here and in all subsequent figures were taken at room temperature using Leica SP1 or SP2 confocal microscopes and software (Version 2.61, build 1537) equipped with 40× and 100× objectives (SP1; PL APO40 × 1.25-0.75NA, PL APO100 × 1.4NA, SP2; HCX PL APO40 × 1.2 NA, PL APO100 × 1.4NA).