Figure 5

Inhibition of IRAG expression by siRNA and effects on calpain activity and relative intracellular Ca²⁺. (a) Suppression of expression of the large form of IRAG by 72 h of incubation after transfection of a mixture of two siRNAs; this reduced the amount of the 150 kDa form of IRAG to ∼30% of control by western blot for total IRAG protein. Changes in the small form of IRAG were not observed, which is expected as the siRNAs targeted the long transcript and the target sequences are not present in the short isoform. This was intentional in that the long form only localizes to the endosomal membranes (see text). (b) When IRAG is suppressed, basal Ca²⁺/calpain is increased, but it is no longer sensitive to NO. Suppression of IRAG increased average cellular μ-calpain activity in the absence of stimulation of PKG (bar 1 versus 4, ^*P<0.05). Preparations incubated 45 min with the strong PKG antagonist Rp-CPT-cGMPS, 50 μM, showed low calpain activity in both control and IRAG-suppressed cells. When sodium nitroprusside was added to IRAG-suppressed or control cells, differences in calpain activity were not significant (bar 2 versus 5). Calpain activity was assayed using t-butoxycarbonyl-Leu-Met-chloromethylaminocoumarin (BOC), which when cleaved by calpain generates a fluorescent signal.² (c) Suppressing IRAG increased average Ca²⁺ in osteoclasts. Calcium was determined using Fluo-3, measuring fluorescence at 520 nM. Fluorescent signal were measured relative to no-cell background at 0, 30, and 45 min, all of which gave similar results. Differences were consistent, with P<0.01 by analysis of variance for control versus IRAG-suppressed cells. Mean±s.e.m., n=7 (control, black) and n=12 (IRAG-suppressed cells, gray).