FIGURE 3.

Strain inhibits GSK3β action through an LRP-independent process. A, Western blot of GSK3β phosphorylation (serine 9) showed an increase at 30 min of strain and a peak response at 1 h. Actin protein was blotted as a control for equal protein loading. B, Western blot of Akt phosphorylation (serine 473) showed a strong increase at 15 min of strain, with the response returning to base line at 1 h. C, strain up-regulation of WISP1 and COX2 expression was not disrupted by treatment with the LRP5 inhibitor Dkk-1 (50 ng/ml). WISP1 and COX2 expression at 4 h of strain were evaluated by real time RT-PCR. Cultures were treated with Dkk-1 1 h prior to strain initiation. The data were normalized to the expression level measured in basal control cells. Data were compiled from three experiments and shown as mean ± S.E. ^*, shows significant effect of strain, p < 0.05. D, Western blots showed that Dkk-1 did not block the increase in phospho-GSK3β (Ser⁹) and phospho-Akt (Ser⁴⁷³) levels by strain. GSK3β was evaluated at 60 min of strain, and Akt was evaluated at 30 min. E, confocal microscopy showed that Dkk-1 did not block strain-induced nuclear translocation of active β-catenin. Active β-catenin level was assessed by immunofluorescence (top) 15 min after strain initiation. Merged images of active β-catenin and nuclear DAPI staining are shown in the bottom.