Using Western blot, GSK3beta phosphorylation at Ser9 is detected in insulin-treated MCF7 cells (+), compared with cells treated with UCN-01 (-), whereas no change in total GSK3beta levels was observed.
Using the GSK3beta assay kits, GSK3beta phosphorylation at Ser9 is detected in insulin-treated MCF7 cells (+), compared with cells treated with UCN-01 (-), whereas no change in total GSK3ß levels was observed.
NIH3T3 cells were seeded at 20K cells/well in medium containing 10% FBS in a 96 well microplate, and cultured for 2 days. The culture medium was removed, and the cells were with varying concentrations of UCN-01 for 2 hours, then stimulated with insulin. The medium was removed from the wells, and the cells were lysed with 120 µL/well Lysis Mix, with shaking for 10 min. 50 µL of lysate was transferred to replicate wells of a PhosphoTracer assay plate, and Antibody Mix specific for either phospho GSK3beta or total GSK3beta (50 µL/well) was added to the lysates. The plates were incubated for 1 hr at room temp, with shaking. Plates were washed and Substrate Mix was added. The plates were covered in foil and incubated for 10 min with shaking. Signal in the wells was determined using a plate reader.