Western blot analysis of Phospho-IKB alpha (S32) on NIH-3T3 cell lysates. Lane 1: NIH/3T3 cells were starved for 18 hours, whole cell lysate, 10 ug/lane. Lane 2: NIH/3T3 cells were starved for 18 hours, then treated with 20 ng/ml TNF alpha for 5 minutes, whole cell lysates, 10 ug/lane. Lane 3: NIH/3T3 cells were starved for 18 hours, then treated with 20 ng/ml TNF alpha for 10 minutes, whole cell lysates, 10 ug/lane. Proteins were transferred to a PVDF membrane and blocked with 5% BSA in PBS for 1 hour at room temperature. The primary antibody Anti-Phospho-IKB alpha (S32) (HA750196, 1/1,000) , Anti-IKB alpha antibody ( ET1603-6, 1/2,000) and Anti-GAPDH antibody (ET1601-4, 1/10,000)was used in 5% BSA at room temperature for 2 hours. Goat Anti-Rabbit IgG H&L (HRP) Secondary Antibody (HA1001) at 1:200,000 dilution was used for 1 hour at room temperature. Predicted band size: 36 kDa Observed band size: 36 kDa Exposure time: 1 minute

Immunocytochemistry analysis of HeLa cells labeling Phospho-IKB alpha (S32) with Rabbit anti-Phospho-IKB alpha (S32) antibody (HA750196) at 1/100 dilution. Cells were fixed in 4% paraformaldehyde for 20 minutes at room temperature, permeabilized with 0.1% Triton X-100 in PBS for 5 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-IKB alpha (S32) antibody (HA750196) at 1/100 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 (M1305-2, 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.