Figure 7.
Hypoxia-induced PKG1 ubiquitination hinders its binding to 8-(2-aminoethyl)thioguanosine-3′,5′ (AET)-cGMP. Cell extracts were prepared from FPASMCs exposed to hypoxia (3% O2, 3 hours) in buffer A, as described in Materials and Methods. (A) The extracts were incubated with 8-AET-cGMP agarose beads. After washing the beads, the bound proteins were eluted with 25 mM cGMP. The total cell lysates, namely, the bead “bound” and “unbound” proteins, were immunoprecipitated with PKG1α antibody and assessed by Western blotting, using an anti-ubiquitin antibody. The blots were stripped and reprobed with PKG1α antibody. (B) The cell extracts were probed for PKG1 and actin. All experiments were repeated 3–4 times. A representative blot is shown. Ubiquitinated PKG1α (Ub-PKG) from hypoxically exposed cells exhibited defective binding to 8-AET-cGMP, compared with native, unmodified PKG1α.