Figure 2

The IP3R-associated cGMP-dependent kinase substrate (IRAG) and the IRAG-binding isoform of PKG1 in osteoclasts. (a) Western blotting for IRAG was performed on anti-IRAG immunoprecipitates from osteoclasts that were untreated (Ctl) or treated with the NO donor sodium nitroprusside (SNP) at 100 μM for 5–10 min, or with the inhibitory cGMP analog 8-(Rp-4-chlorophenylthio)guanosine-3′,5′-cyclic phosphorothioate (Rp-cGMPS), 50 μM, for 30 min with or without subsequent treatment with SNP (100 μM for 10 min). Western blotting showed similar amounts of the large and small forms of IRAG, with Mr of ∼150 and 100 kDa, under all conditions. The last lane shows an isotype control (nonimmune serum). (b) Anti-phosphoserine blot (upper panel) of anti-IRAG immunoprecipitates from untreated osteoclasts (Ctl) or osteoclasts treated with 100 μM SNP for 5–10 min, or with 50 μM Rp-cGMPS for 30 min. The blots were then stripped and reprobed with anti-IRAG antibody to confirm similar recovery of IRAG (lower panel). IRAG phosphoserine was increased after treatment with SNP, but reduced by the PKG antagonist. (c) Relative expression of PKG1 isoforms in osteoclasts. Quantitative PCR using primers specific to PKG1β or PKG1α or recognizing both forms (Common) was performed. The amount of product for each primer set is shown relative to GAPDH as an internal control. The amount of PKG1β was indistinguishable from total PKG1. PKG1α mRNA was present, but at levels significantly lower than PKG1β (^*P<0.05; NS, not significant).