Figure 4

Effects of NO donors on distribution of IRAG at non-endosomal sites. (a) At the plane of cell attachment to glass substrate, antibody reacting with long and short forms of IRAG (red) did not localize with punctate cellular attachments (top, phalloidin, green; arrows). However, after NO donor activation, IRAG localized in cellular attachments (bottom, yellow; arrows). Fields are 30 μm². IP3R1 precipitates from osteoclasts treated or not with NO donor or PKG inhibitor and IRAG precipitates from supernatants after IP3R1 precipitation were blotted for migfilin and VASP (bottom panels). In IP3R1 precipitates (lanes 1–3) there was no significant migfilin. After depletion of IP3R1, precipitates of supernatant IRAG included migfilin. This association was increased by sodium nitroprusside. This fraction also included VASP, which also increased with sodium nitroprusside. VASP in IP3R1 precipitates was not determined. (b) In cells treated with NO donors, IRAG nuclear localization was increased relative to cells in which PKG was blocked. Similar effects were observed using activating cGMP analogs (not illustrated). Fields are 50 μm². (c) Effect of PKG knockdown on redistribution of IRAG to nuclei. PKG siRNA reduced PKG expression by approximately 80% (top); the amount of PKG was not affected by sodium nitroprusside (100 μM treatment, 30 min before cell lysis). In PKG-silenced cells (siRNA shown by green label), after sodium nitroprusside treatment (100 μM, 10 min), IRAG was no longer redistributed to nuclei (bottom left), whereas in cells with control siRNA, also after sodium nitroprusside treatment, nuclear localization was strong (bottom right).