PKCζ activation by d-flow and ONOO⁻. (A and B) Generation of s-flow and d-flow using a cone and plate flow chamber. Tracks of fluorescent beads in a cone and plate flow chamber. S-flow was generated using a nongrooved cone (A), and d-flow was generated using a grooved cone (B). Note straight tracks with s-flow and short tracks with different orientations with d-flow, which are caused by beads going out of focus. Both cones were rotated at the same speed. Color tracks indicate time x (red), time x + 10 s (green), and time x + 20 s (blue). Bright dots are beads adhered to the dish. Exposure time is 0.4 s. Bars, 100 µm. (C and D) HUVECs were stimulated by s- or d-flow (C) or ONOO⁻ (D) for the indicated times, and PKCζ phosphorylation was determined by Western blotting. The level of PKCζ phosphorylation was determined by taking the ratio of optical densities between antiphospho-PKCζ and anti-PKCζ bands (bar graphs) as described in Materials and methods. The experiments were performed in triplicate using three different batches of s- or d-flow or ONOO⁻-stimulated HUVECs (means ± SD; n = 3; *, P < 0.05; and **, P < 0.01 compared with control). Molecular masses are given in kilodaltons. IB, immunoblot.