Effect of siRNA depletion of RAB1B and RUNX1 on vWF trafficking in HEL cells. (A) PMA-treated HEL cells were transfected with siRNAs and expression plasmid, as indicated and as described in the legend for Figure 4. In control siRNA-transfected cells, vWF (green) colocalized with the Golgi (E2-Crimson-GalT, red; Pearson correlation coefficient r = 0.793 ± 0.018). On treatment with RUNX1 siRNA (r = 0.478 ± 0.072; P < .05; compared with control) or RAB1B siRNA (r = 0.429 ± 0.082; P < .05), Golgi structures were disrupted and the colocalization with vWF was lost. RAB1B expression in RUNX1 siRNA-depleted cells reconstituted the Golgi structures along with colocalization of vWF (r = 0.665 ± 0.082; P = NS), similar to that seen in control cells. Bar, 10 μm. (B) Immunoblot analysis of vWF, RUNX1, RAB1B, and actin in HEL cells treated as described earlier. Downregulation of RAB1B or RUNX1 decreased vWF; reconstitution of RAB1B in RUNX1-depleted cells restored the vWF. (C) Platelet vWF by immunoblotting in 3 patients with RUNX1 mutations and 5 normal subjects. Also shown is the ratio of vWF to GAPDH as loading control.