spacer gif spacer gif spacer gif spacer gif spacer gif
QUICK SEARCH:   [advanced]


spacer gif
     Home     Help     Feedback     Subscriptions     Archive     Search     Table of Contents    

Right arrow Help viewing high resolution images
Right arrow Return to article
(Downloading may take up to 30 seconds.
If the slide opens in your browser, select File -> Save As to save it.)
Click on image to view larger version.


Figure 4


Fig. 4. RGS10A signaling is essential for induction of [Ca2+]i oscillations by RANKL and NFAT2 expression. (A) [Ca2+]i oscillations were observed in RANKL-stimulated BMMs or RAW264.7 cells, 48-72 hours after RANKL stimulation. The picture shows an example of successive pseudocolored Ca2+ images of cells treated with RANKL for 48 hours. Oscillations were produced at 2-minute intervals. (B) Ca2+ changes were traced in single RGS10A-silenced or control cells treated with RANKL and M-CSF for 72 hours. Ca2+ changes were estimated as the ratio of fluorescence intensity of the Fluo-4 to Fura Red, plotted at 5-second intervals. Each color indicates a different cell in the same field. [Ca2+]i oscillations are blocked in RGS10A-silenced cells (pAVU-R10a). (C) Immunofluorescence revealed that the expression of NFAT2 was blocked in RGS10A-silenced cells. (D) There were weak signals of NFAT2 protein detected in RGS10A-silenced cells (lane 3) as compared to the controls (lane 1: mock; lane 2: pAVU6). (E) Quantification of the bands in D. NFAT2 signals in the mock or pAVU6, are 4-4.7 times stronger than in pAVU-R10a. (F) Western blot analysis of activation of PLC{gamma} after stimulation with RANKL for 40 minutes. Phosphorylation of PLC{gamma} was impaired in the RGS10A-silenced group (plenty-R10a) following a 40-minute stimulation with RANKL. (G) Co-immunoprecipitation of RGS10A and CaM. No interaction was observed in unstimulated cells. CaM bound to RGS10A in the presence of 1 mM CaCl2. This interaction was blocked by 0.5 mM EGTA. (H) PdIns(3,4,5)P3-bead binding assay. RGS10A was not detected after stimulation with M-CSF alone (lane 1) or with control beads without PdIns(3,4,5)P3 (negative control, lane 2). RGS10A was detected after stimulation with RANKL (positive control, lane 3). RGS10A was detected after stimulation with RANKL in a PdIns(3,4,5)P3 pull-down assay (lane 4). (I) mRNA expression of RGS 2, 4, 5, and 10A in RANKL-stimulated OCLs. RGS10A is predominantly expressed in OCLs, compared with RGS2, RGS4, and RGS5.





Right arrow Return to article