Immunocytochemistry analysis of HeLa cells untreated / treated with 100nM Calyculin A and 20ng/mL TNF-α for 15 minutes labeling Phospho-NF-kB p65 (S536) with Rabbit anti-Phospho-NF-kB p65 (S536) antibody (HA751361) at 1/500 dilution. Cells were fixed in 4% paraformaldehyde for 15 minutes at room temperature, permeabilized with 0.1% Triton X-100 in PBS for 15 minutes at room temperature, then blocked with 1% BSA in 10% negative goat serum for 1 hour at room temperature. Cells were then incubated with Rabbit anti-Phospho-NF-kB p65 (S536) antibody (HA751361) at 1/500 dilution in 1% BSA in PBST overnight at 4 ℃. Goat Anti-Rabbit IgG H&L (iFluor™ 488, HA1121) was used as the secondary antibody at 1/1,000 dilution. PBS instead of the primary antibody was used as the secondary antibody only control. Nuclear DNA was labelled in blue with DAPI. Beta tubulin (HA601187, red) was stained at 1/100 dilution overnight at +4℃. Goat Anti-Mouse IgG H&L (iFluor™ 594, HA1126) was used as the secondary antibody at 1/1,000 dilution.

Western blot analysis of Phospho-NF-kB p65 (S536) on different lysates with Rabbit anti-Phospho-NF-kB p65 (S536) antibody (HA751361) at 1/5,000 dilution. Lane 1: C6 cell lysate (20 µg/Lane) Lane 2: C6 treated with 100ng/mL Calyculin A for 1 hour cell lysate (20 µg/Lane) Lane 3: PC-12 cell lysate (20 µg/Lane) Lane 4: PC-12 treated with 100nM Calyculin A for 30 minutes cell lysate (20 µg/Lane) Predicted band size: 60 kDa Observed band size: 65 kDa Exposure time: 59 seconds; ECL: K1801; 4-20% SDS-PAGE gel. Proteins were transferred to a PVDF membrane and blocked with 5% NFDM/TBST for 1 hour at room temperature. The primary antibody (HA751361) at 1/5,000 dilution was used in primary antibody dilution (K1803) at 4℃ overnight. Goat Anti-Rabbit IgG - HRP Secondary Antibody (HA1001) at 1/50,000 dilution was used for 1 hour at room temperature.