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Fig. 6. Src activity is required for calpain activation by cGMP, but levels of phosphorylated Akt are not increased. (A) Src is required for cGMP-mediated activation of calpain in osteoclasts. These assays used the BOC fluorescent calpain substrate as described in Fig. 1. In the absence of cGMP (untreated) there is weak basal activity, attributed to autocrine NO production. Following activation with 8-Br-cGMP (50 µM, 1 hour) calpain is strongly activated. The Src inhibitor PP2 reduced activity of calpain to below that of the control (PP2) and reduced 8-Br-cGMP (cGMP) activation by 85% (PP2+8-Br-cGMP). An inactive control, PP3, had no effect on 8-Br-cGMP (cGMP) activation (PP3+8-Br-cGMP). *P<0.01 relative to 8-Br-cGMP; n=25-60, mean ± s.e.m. (B) Activation of cGMP did not increase phosphorylation of Akt. Phosphorylated Akt (p-Akt) was identified in osteoclast lysates by antibody against Akt phosphorylated at Ser473. Lysates of cultures after treatment with the hydrolysis-resistant cGMP analog 8-pCPT-cGMP (100 µM) or the cGMP-blocking analog Rp-cGMPS (50 µM) for 30 minutes were compared with untreated cells. Density of antibody labeling was measured and expressed as a fraction of total Akt by stripping membranes and re-labeling for total Akt protein. Whereas no differences were significant, the largest p-AKT signal was in the control containing the cGMP-blocking analog (left bar). Since PI 3-kinase activates Akt phosphorylation, this suggests that PI 3-kinase is not a direct mediator of cGMP signaling in osteoclasts; n=3, mean ± s.e.m. (C) PKG1 and VASP are required for cGMP-dependent Src phosphorylation. Phosphorylation of Src (top) at Y416 after treatment with 100 µM 8-pCPT-cGMP was reduced by knockdown of PKG1 (left) or VASP (right). Protein loading controls (middle) and knockdown controls (bottom) are also shown. In either case, p-Src was reduced by 70-90%. siRNAs used for knockdown experiments have been described previously (Yaroslavskiy et al., 2005).
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