FIGURE 1.

Effect of unidirectional laminar shear on nuclear levels of the NFkB subunits p50 and p65 and binding of these to the eNOS promoter. Western blots are shown of nuclear extracts for p50 (A) and p65 (B). C shows mean densitometry analysis of three to four separate experiments for each. Data are mean ± S.E. of the mean. D shows a Western blot for the cytoplasmic protein α-tubulin and α-actin (present in both the nucleus and cytoplasm) from a nuclear preparation (lane 1) and a cytoplasmic preparation (lane 2). E shows ChIP analysis of binding of p50 and p65 to the eNOS promoter. P50 and p65 were immunoprecipitated from nuclear extracts and segments of eNOS DNA amplified by PCR. Input refers to nuclear extracts before immunoprecipitation. IgG was used as an immunoprecipitation control. F shows mean values for densitometric analysis of three similar experiments. As evident, shear only increased nuclear levels of p50 and p65 by 2-fold but dramatically increased binding of these subunits to the endogenous eNOS promoter.