Western blot analysis of lysates from NIH/3T3 cells, using Phospho-ERK1-T202 + ERK2-T185 Rabbit mAb (AP0485) at 1:1000 dilution. NIH/3T3 cells were treated with PMA/TPA (200 nM) at 37℃ for 30 minutes after serum-starvation overnight.
Secondary antibody: HRP-conjugated Goat anti-Rabbit IgG (H+L) (AS014) at 1:10000 dilution.
Lysates/proteins: 25μg per lane.
Blocking buffer: 3% nonfat dry milk in TBST.
Detection: ECL Basic Kit (RM00020).
Exposure time: 1S.