Figure 3

Localization of IRAG at endosomal sites. Photomicrographs of human osteoclasts on glass coverslips with immune labeling as indicated. Antibody N19 (see Materials and methods) recognizing the 150 kDa form of IRAG was used. To determine colocalization of IP3R1 without subjectivity, labeling of pixels for both IRAG and IP3R1 were calculated by digital coincidence selection (see Materials and methods); the results are shown in black in columns labeled Coincidence. Colocalization was more prominent when PKG was inhibited by Rp-cGMPS, than when PKG was activated by NO, and this difference was accentuated when Ca²⁺ was low. All fields shown are 50 μm². (a) IP3R1 and IRAG in human osteoclasts on glass coverslips with PKG inhibited by 50 μM Rp-cGMPS (30 min of incubation; top row of panels) or activated by 100 μM sodium nitroprusside (10 min of incubation, bottom row of panels). (b) The same experiment was performed after pre-treatment with the cell-permeant Ca²⁺ chelator-donor, 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetate (BAPTA, 70 μM, as acetoxymethyl ester) added 40 min before the PKG effectors.