Fig. 6.

BzATP induces inhibitory phosphorylation of GSK3α/β. a MC3T3-E1 osteoblast-like cells were seeded at a density of 1.5 × 10⁴ cells/cm² in a 6-well plate and cultured for 1 day. Cells were then placed in serum-free media and incubated overnight. The next day, cells were treated under serum-free conditions with vehicle (V) or BzATP (300 μM; BzATP) for the times indicated, and total protein was harvested. Samples were subjected to immunoblot analyses using specific pSer21/9 and pTyr279/216 GSK3α/β antibodies. As a loading control, blots were also probed for total GSK3α/β. Bands were visualized by the ECL method. Images are representative blots from three to five independent preparations. b pSer21/9, pTyr279/216, and total GSK3α/β bands were evaluated by densitometry. The ratio of pGSK3α/β to total GSK3α/β was determined for serine and tyrosine residues, and results were normalized to vehicle. Treatment with BzATP had no significant effect on the phosphorylation status of tyrosine 279/216 (quantification not shown). α indicates a significant difference from 0 min (p < 0.05). Data are means ± SEM (n = 3–4 independent preparations)