Figure 6. PTEN deficiency in vivo promotes loss of SRF binding to SM gene promoters.

(a) PTEN was immunoprecipitated from WCL of smooth muscle-rich intact aortic media of wild-type mice. About 10% of input WCL and co-immunoprecipitating SRF were detected by immunoblotting. Representative blot showing co-IPs from two separate mice; N=6 independent mice analysed. *, heavy chain IgG. (b) ChIP assays for PTEN and SRF binding to CArG elements in the Myh11 (left) and Fos (right) promoters were performed on intact aortic media using pooled arteries from five individual wild-type mice. (c–e) Mice underwent carotid artery ligation-induced injury as described in Methods. Chromatin was isolated from 48-h injured and contra-lateral uninjured arteries and analysed by ChIP for SRF (c–e) and PTEN (c,d) binding to CArG elements in the Myh11 (c), Acta2 (d) and Fos (e) promoters. Data represent average fold changes±s.e.m. N=3 independent experiments using pooled arteries from 9 to 11 individual mice; *P<0.01 versus uninjured control; ‘**' denotes statistical analysis not conducted due to complete loss of PTEN interactions in injured vessels (consistent undetectable values). (f–h) Smooth muscle-rich aortic media from wild-type (WT) and PTEN iKO (iKO) mice was analysed by ChIP for SRF binding to CArG elements in the Myh11 (f), Acta2 (g) and Fos (h) promoters. Data represent average fold changes±s.e.m. N=3 independent experiments using pooled arteries from five individual mice; *P<0.01 versus WT. (i) Western blot analysis for c-fos levels in whole cell lysates (WCL) of aortic media from WT or PTEN iKO mice. β-Actin was used as a loading control (western blot is from the same samples shown in Fig. 1c using the same β-Actin image). Representative blot from two mice per genotype with each lane representing an individual mouse. Molecular weight markers were cropped out for final SRF blot; please see Supplementary Fig. 8.