Figure 2
GRIP1 in the EFNB3 reverse signaling pathway in VSMCs.
Experiments in this figure were repeated more than twice, and representative data are shown. (A) Effective mRNA knockdown of Grip1 by siRNA. VSMCs from female WT mice were transfected with siRNAs of a particular gene or control siRNA, as indicated. After 24-h culture, the cells were harvested and the mRNA expression of each gene was determined by RT-qPCR. The data are expressed as means ± SD of the ratios of the target gene signal versus the β-actin signal. The data were analyzed by one-way Student’s t test. *p < 0.05. (B) GRIP1 knockdown by siRNAs reverses the effect of solid-phase anti-EFNB3 Ab on reducing VSMC contractility. VSMCs from female WT and KO mice were cultured in wells coated with goat anti-mouse EFNB3 Ab (2 μg/ml during coating). After 2 days, they were transfected with siRNAs targeting Grip1, or with control siRNA. On day 4 of culture, they were stimulated with PE (20 μmol/L), and their percentage contraction was recorded. Means ± SD of the percentage are reported. The thin line represents mean contractility of VSMCs cultured in well without coating of anti-EFNB3 Ab as an additional control; for better viewing, SD was not added to the line. The data were analyzed with one-way ANOVA followed by ad hoc analysis, and p-values of significant difference are indicated. (C) Grip1 siRNA in the absence of EFNB3 reversing signaling had no effect on VSMC contractility. VSMCs from female WT mice were cultured in plain wells without Ab coating. They were transfected with Grip1 or control siRNA and then stimulated with PE as described in (A). Mean ± SD of percentage contraction are shown. The data were analyzed with one-way ANOVA but not statistically significant difference between the test and control groups is found.
