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Fig. 4. Western blot studies of VASP in human osteoclasts, VASP-associated proteins in VASP precipitates, the effect of PKG I suppression on VASP phosphorylation, and the time-course of VASP phosphorylation after cGMP or NO activation. (A) Osteoclast lysates probed for VASP. VASP is abundant and occurs in cGMP-inhibited (Ctl, 50 µM Rp-cGMPS) and cGMP-stimulated (cGMP, 100 µM 8-pCPT-cGMP) osteoclasts in similar amounts. Fifteen µg lysates of osteoclasts were separated on 10% polyacrylamide in Laemmli buffer, and transferred to PVDF for labeling. Std, 100 ng of recombinant VASP. (B) VASP immunoprecipitates in cGMP-inhibited and -activated osteoclasts, probed for migfilin, 
v integrin and VASP. Immunoprecipitation for VASP was done using 900 µg lysates of osteoclasts after treatment with 50 µM Rp-cGMPS for 1 hour (Inhib) or with 100 µM 8-pCPT-cGMP for 1 hour (cGMP). These conditions essentially eliminate (Rp-cGMPS) or strongly promote (8-pCPT-cGMP) PKG I activity. Note that VASP is phosphorylated by activated PKG I. This also changed the relative abundance of its associated proteins. Migfilin was co-precipitated with VASP mainly in cGMP-activated cells (top panel), whereas v integrin precipitation by anti-VASP was reduced by cGMP. Phospho-Vasp (p-VASP), as expected, was greatly increased by cGMP activation relative to VASP. Two or more blots gave similar results. (C) Western blots of PKG I, phospho-VASP, VASP and actin in human osteoclasts after siRNA transfection targeting PKG I relative to mock-transfected control cells. Each lane is a 15 µg osteoclast lysate separated as in A, using cells either transfected with siRNA targeting PKG I as in Fig. 3 (left lane), or a mock-transfected control (right lane). The efficiency of siRNA targeting (
75%) was lower than in the motility study shown in Fig. 3, but nonetheless it markedly reduced phospho-VASP relative to the mock-transfected control cells. (D) Phospho-VASP in VASP immunoprecipitates as a function of time after treatment of osteoclasts with 8-pCPT-cGMP or S-nitroso-N-acetylpenicillamine. Polyclonal anti-VASP was used for immunoprecipitation of protein from 900 µg osteoclast lysates, either without treatment or after 100 µM 8-pCPT-cGMP or 60 µM S-nitroso-N-acetylpenicillamine for the times indicated. Each lane (except an isoimmune control for immunoprecipitation by the antibody and a medium control with no cell lysate) represents an osteoclast immunoprecipitate separated on 9% SDS-PAGE as in A, with immunolabeling using antibody to phospho-VASP (p-VASP) compared to total VASP. Phospho-VASP was detected using anti phospho-Ser239-VASP monoclonal antibody. Increases in phospho-VASP using 8-pCPT-cGMP typically peaked by 1 hour, whereas S-nitroso-N-acetylpenicillamine (SNAP, lower left) peaked at 2-3 hours, in keeping with the half-life of that agent. Sodium nitroprusside gave a similar response but p-VASP increased within 10 minutes in keeping with the short half life of sodium nitroprusside (not illustrated).
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