Figure 5.

Src251 osteoclasts have reduced AKT kinase activity. (A) Osteoclasts were differentiated from bone marrow in culture, then lysed, immunoprecipitated with anti-AKT serum, and in vitro kinase assays were performed using GST–GSK3α as a substrate. Products were detected using an anti-phospho GSK-3 antibody by immunoblotting. After normalization for AKT protein levels, AKT activity was observed to be decreased twofold. In this experiment, twofold lower levels of AKT protein were also observed. (Lower panel) Lysates were normalized by immunoblotting for TRAP protein. (B) Osteoclasts were lysed as above and TRAP-normalized lysates were immunoblotted with anti-active ERK. Equivalent levels of total ERK were observed in these samples.