P-selectin expression in HUVECs is regulated by p38 kinase pathways. (A) Phosphorylation of p38 kinases increases in HUVECs treated with hypoxia for 1 hour with or without the NOS inhibitor l-NAME (100 μM). A representative blot was shown from 3 independent experiments. (B) P-selectin expression on the cell surface of HUVECs is regulated by p38 kinases. HUVECs were treated with anisomycin (100 ng/mL) (upper panel) or SB203580 (5 μM) (lower panel) for 1 hour, and P-selectin expression on the cell surface was evaluated by immunofluorescence microscopy as described in “Materials and methods.” MFIs were calculated by scanning fluorescence images with ImageJ v1.43 (NIH). (C) Knockdown of p38 kinase expression in HUVECs by siRNA constructs. Western blot showing that p38 siRNA, but not control negative siRNA, decreased total p38 protein by 50% to 66% in HUVECs at 48 hours. (D) P-selectin expression in HUVECs transfected with siRNA constructs for p38α kinase (gray columns) and negative control siRNA (black columns). Expression of P-selectin on the cell surface of HUVECs was quantitated by immunofluorescence microscopy as described in “Materials and methods,” and MFIs were calculated by scanning fluorescence images with ImageJ v1.43 (NIH).