Western blot analysis of extracts of various cell lines, using Phospho-MEK1-T286 antibody (TA378246) at 1:2000 dilution or MEK1 antibody (A12687). HeLa cells were treated by nocodazole (50 ng/mL) at 37°C for 20 hours or treated by ATP(5 mM) at 30°C for 1 hour. NIH/3T3 cells were treated by Nocodazole (50 ng/mL) at 37°C for 20 hours or treated by ATP(5 mM) at 30°C for 1 hour. PC-12 cells were treated by Nocodazole (1 microg/mL) at 37°C for 16 hours or treated by ATP(5 mM) at 30°C for 1 hour. - Secondary antibody: HRP Goat Anti-Rabbit IgG (H+L) at 1:10000 dilution. - Lysates/proteins: 25ug per lane. - Blocking buffer: 3% BSA. - Detection: ECL Basic Kit . - Exposure time: 30s.