Western blot analysis of extracts of various cell lines, using Phospho-NF-kB p65/RelA-S529 antibody (TA380848) at 1:1000 dilution.HeLa cells were treated by both TNF-α(20ng/mL) and Calyculin A (100nM) for 10 minutes.NIH/3T3 cells were treated by TNF-α(20ng/mL) for 30 minutes.|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: 1s.