Figure 4.

Inhibitory effect of Rab35 knockdown on histamine-evoked VWF secretion. A and B, HUVEC were transfected twice for 48 h with a Rab35-specific siRNA and a nonspecific siRNA, which served as negative control. A, Western blot analysis of Rab35-depleted cells, which were co-transfected with wild-type Rab35–GFP. The knockdown efficiency was determined by immunoblotting of total cell lysate using anti-GFP antibodies as well as anti-Rab35 antibodies to probe for the endogenous protein. α-Tubulin was used as loading control. IB, immunoblot. B, VWF secretion levels in Rab35-depleted cells. HUVEC were treated with histamine-containing stimulation medium and processed for ELISA-based secretion analysis as described under “Experimental procedures.” Results are expressed as the mean ± S.E. (error bars) of 12 independent experiments (***, p < 0.001; paired t test).