FIGURE 6.

Grip1 in the Efnb reverse signaling pathway in VSMC. The experiments in this figure were repeated more than twice, and representative data are shown. A, effective mRNA knockdown of Disheveled, PDZ-RGS3, and Grip1 by siRNAs. Cultured WT VSMC were transfected with a mixture of siRNAs of a particular gene or control siRNAs, as indicated. After additional 24-h culture, the cells were harvested, and the mRNA expression of each gene was determined by RT-qPCR. The data are expressed as the means ± S.D. of the ratios of the target gene signal versus the β-actin signal. B, Grip1 knockdown by siRNAs partially reversed the inhibitory effect of solid phase Ephb6-Fc. VSMC from WT males were cultured in wells coated with Ephb6-Fc (2 μg/ml for coating). After 2 days, the cells were transfected with siRNAs targeting Disheveled, PDZ-RGS3, or Grip1 or with control siRNA. On day 4 of culture, they were stimulated with PE (20 μm), and their percentage contraction was registered. Means ± S.D. of the percentage are shown. The thin line (indicated as Normal Contraction) represents the mean percentage contraction of VSMC cultured in wells coated with normal human IgG (2 μg/ml) without siRNA transfection (for a better visual effect, the S.D. of each time point in this control is omitted). *, p < 0.05 according to paired Student's t test, between Grip1 siRNA- and control siRNA-transfected VSMC.