Western blot analysis of extracts of various cell lines, using Phospho-AKT1-S473 pAb (TA373415) at 1:1000 dilution.NIH/3T3 cells were treated by Calyculin A (100 nM) at 37‚ÑÉ for 30 minutes after serum-starvation overnight.|Secondary antibody: HRP Goat Anti-Rabbit IgG (H+L) at 1:10000 dilution.|Lysates/proteins: 25ug per lane.|Blocking buffer: 3% nonfat dry milk in TBST.|Detection: ECL Basic Kit .|Exposure time: 10s.