KD of Rab27A, but not of Rab3B or Rab3D, decreases hormone-evoked VWF secretion. (Ai-ii) Quantification of mRNA and protein after nucleofection with either control (siCTRL) or specific KD oligos as indicated. mRNA KD were Rab3B (89% ± 2%), Rab27A (92% ± 0.4%), Rab3B/Rab27A (81% ± 1%/87% ± 2%), and Rab3D (87% ± 2.6%). Data were pooled from 3-5 independent experiments. Representative immunoblots are shown. Position of molecular weight markers (kDa) are shown to the right; α-tubulin (Ai) or transferrin receptor (TfR, Aii) were used as loading controls. (Aiii) Four left panels: Immunostainings of HUVECs for endogenous Rab3B (mouse Ab, green) or Rab27A (mouse Ab, green) and VWF (sheep Ab, red) in control or KDs as indicated. Two right panels: Immunostainings of HUVECs for Rab3B (mouse Ab, green), Rab27A (rabbit Ab, blue), and VWF (sheep Ab, red). Grayscale images are from regions indicated by white boxes. Scale bars represent 20 μm. (Aiv) Immunostainings of HUVECs for Slp4-a (rabbit Ab, green in color merge on left) and VWF (sheep Ab, red in color merge on left) in control (top) or Rab3B/Rab27A KD (bottom). Scale bars represent 20 μm. (B) Histamine-evoked VWF secretion from HUVECs nucleofected with control or specific siRNA oligos for Rab3B, Rab27A, or Rab3B/Rab27A (Bi), or Rab3D (Bii) as indicated. Open bars represent control (vehicle alone); and gray bars, histamine stimulation. Data were pooled from 3 independent experiments. There was no significant difference in VWF secretion between Rab27A KD and Rab3B/Rab27A KD (P = .26). Compared with siCTRL, basal secretion of VWF was significantly lower in Rab27A (P = .02) or Rab27A/Rab3B (P = .015) KD experiments.