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. 2016 Mar 4;7:10830. doi: 10.1038/ncomms10830

Figure 2. PTEN forms a nuclear multi-protein complex with SRF and myocardin.

Figure 2

(a,b) PTEN (top) or SRF (bottom) proteins were immunoprecipitated (IP) from WCL of cultured human (a) or rat (b) aortic SMCs serum restricted for 48 h. About 10% of input WCL and co-immunoprecipitating SRF or PTEN were detected by immunoblotting (IB). A non-specific IgG was used for IPs as a negative control. (c) SMCs stably expressing control (Ctrl) or PTEN-specific shRNA were transiently transduced with empty vector adenovirus (EV) or adenoviruses encoding wild-type PTEN (WT) or phosphatase-inactive PTEN (MT) (multiplicity of infection=100). PTEN was immunoprecipitated (IP) and co-immunoprecipitating SRF was detected by immunoblotting (IB). (d) Cytoplasmic (cyto) and nuclear (nuc) SMC extracts were analysed by western blotting for SRF and PTEN levels. β-Tubulin and Lamin A/C were used as cytoplasmic and nuclear loading controls, respectively. (e) PTEN was immunoprecipitated from cytoplasmic and nuclear SMC extracts and co-immunoprecipitating SRF was detected by immunoblotting. A non-specific IgG was used for IPs as a negative control. (f) Recombinant His-tagged PTEN and GST-tagged SRF were purified, incubated together, and PTEN (top) or SRF (bottom) were immunoprecipitated. Co-immunoprecipitating SRF or PTEN was detected by immunoblotting. About 10% of protein mixture was immunoblotted to control for input. A non-specific IgG was used for IPs as a negative control. (g) Myocardin (left), Elk-1 (middle) and SRF or PTEN (right) proteins were immunoprecipitated from WCL of SMCs serum restricted for 48 h. About 10% of input WCL and co-immunoprecipitating SRF, PTEN or Elk-1 were detected by immunoblotting. Shown are representative blots from a minimum of three independent experiments. *, heavy chain IgG. Molecular weight markers were cropped out for final SRF blots; please see Supplementary Fig. 8.