Western blot analysis of various lysates, using Phospho-NF-kB p65/RelA-S536 Rabbit mAb (AP1294) at1:1000 dilution (upper) or [KO Validated] NF-kB p65/RelA Rabbit mAb (A22331) at1:50000 dilution (lower) incubated overnight at 4℃. HeLa cells were treated by Calyculin A (100 nM) at 37℃ for 30 minutes after serum-starvation overnight.C6 cells were treated by TNF-α (20 ng/ml) and Calyculin A (50 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: 30 μg per lane.
Blocking buffer: 3% nonfat dry milk in TBST.
Detection: ECL Basic Kit (RM00020).
Exposure time: 10s.