Fig 2: Conditional SMC-specific FXR1 knockout mice are hypotensive(A) Conditional expression of FXR1. Immunohistochemistry of FXR1 protein in conditional FXR1SMC/SMC mice following 5 days of intraperitoneal (i.p.) tamoxifen injections (80 μg/kg/day) or corn oil. Figure shows FXR1 expression (red-brown staining) only in endothelium and adventitia of tamoxifen-injected mice.(B) Western blot from gastroc and aorta showing differing FXR1 expression following tamoxifen injections.(C) Conditional FXR1smc/smc mice have decreased diastolic and mean arterial blood pressure compared with controls. Telemetric measurements were taken for 26 h. **p < 0.001. WRC, GTPase, and cytoskeleton-associated gene expression are reduced in aorta from FXR1SMC/SMC.(D) RNA isolated from aorta from mice injected with oil or tamoxifen were subjected to qRT-PCR to validate FXR1 knockout.(E and F) Rac1 mRNA and protein are significantly decreased in aorta from tamoxifen-injected compared with control mice.(G and H) CYFIP1 mRNA and protein are significantly decreased in aorta from tamoxifen-injected compared with control mice. Western blot shown is representative of at least 3 experiments performed from aorta from at least 3 mice.*p < 0.05, **p < 0.01, and ***p < 0.001 from at least three aortas.

Fig 3: FXR1 knockdown reduces WAVE mRNA abundance, mRNA stability, and protein abundancehVSMCs were transfected with control or FXR1 siRNA, stimulated with PDGF-AB for the times noted, and total mRNA determined by qRT-PCR. To determine mRNA stability, samples were stimulated with PDGF-AB for 16 h, actinomycin D added, RNA isolated at the indicated time points, and mRNA quantified by RT-PCR. For RIP-PCR, transcripts were recognized and pulled down by FLAG-tagged FXR1 adenovirus, reverse transcribed, and amplified by PCR. For protein abundance, protein was isolated from lysates from hVSMCs transfected with control or FXR1 siRNA and detected by western blot. Densiometric analysis was determined from 3–5 independent experiments.(A) Arp2 RNA abundance, stability, and mRNA interaction by RIP-PCR.(B) Arp2 protein abundance.(C) CYFIP1 RNA abundance, stability, and mRNA interaction by RIP-PCR.(D) CYFIP1 protein abundance. Representative blots are shown of at least 3 independent experiments performed.*p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001 from at least three experiments.