Figure 3.

Knockdown of kinesin-1 blocks TEM. A: Western blot analysis was performed 72 hours after infection with scrambled shRNA, kinesin-1 shRNA (KHC shRNA), or kinesin-1 shRNA with addition of the kinesin-1 rescue construct tagged with mCherry (upper band in KHC shRNA + Rescue lane). A polyclonal antibody against kinesin-1 was used to probe for KHC. Quantification of the relative total KHC expression. B: After knockdown of kinesin-1, cells were stained for VE-cadherin, PECAM, and microtubules. Insets show enlargements of the area in dotted boxes. C and D: PBMCs were allowed to transmigrate on infected HUVEC monolayers for 8.5 minutes before quantifying adhesion (C) and migration to cell borders (D). E: PBMCs were allowed to transmigrate on HUVEC monolayers for 60 minutes to measure TEM. Data are expressed as means ± SEM for all experiments. n = 3 experiments each with three monolayers/variable per experiment with a total of >600 cell interactions counted (C–E). ^∗P < 0.05, ^∗∗P < 0.01. Scale bar = 10 μm. Original magnification, ×312 (insets). HUVEC, human umbilical vein endothelial cell; KHC, kinesin heavy chain; PBMC, peripheral blood mononuclear cell; PECAM, platelet-endothelial cell adhesion molecule; TEM, transendothelial migration; VE, vascular endothelial.