FIG 3.

Notch promotes a switch in Akt isoform expression, inactivates GSK-3β, and accumulates Snail1 in the nucleus. (A and C) The indicated proteins were analyzed by Western blotting in total (A) or cytosolic (lanes CE) and nuclear (lanes NE) extracts (C). Tubulin and lamin B were used as a control for cytosolic and nuclear compartments, respectively. (B) pS9-GSK-3β levels were quantified by densitometric analysis and normalized respect to total GSK-3β; the averages ± the SD (n = 4) are shown (*, P < 0.05). (D) Active levels of Akt1 and Akt2 were determined after immunoprecipitation (IP) of both isoforms, which were blotted against the respective phosphorylated proteins. Irr Ab, irrelevant control antibody; IgG, immunoglobulin band. (E) pS9-GSK-3β levels were analyzed by immunofluorescence in PAE-CTL and PAE-NICD2 cells (red); DAPI was used to identify nuclei. (F) PAE cells stably transfected with NICD-myc (NICD2) or empty vector (CTL) were treated with TGF-β1 for 24 h, and the indicated proteins were analyzed by Western blotting.