710676

antibody from Invitrogen Antibodies

Targeting: IKBKB IKK-beta, IKK2, IKKB, NFKBIKB

Immunocytochemistry (Supportive data in Antibodypedia)

Antibody Data

Product number

710676

Provider

Invitrogen Antibodies

Product name

Phospho-IKK alpha/beta (Ser176, Ser180) Recombinant Polyclonal Antibody (7HCLC)

Antibody type

Polyclonal

Antigen

Synthetic peptide

Description

This antibody is predicted to react with Dog, Rabbit, Pig, Mouse, Rat, Bovine and Goat.

Antibody clone number

7HCLC

Concentration

0.5 mg/mL

Immunocytochemistry

Supportive validation

Submitted by

Invitrogen Antibodies (provider)

Main image

Experimental details

Time course showing induction of the TNF-a signaling cascade upon treatment: Cellular localization of proteins in the NF-KB signaling pathway was detected upon treatment of HeLa cells with TNF alpha (50 ng/mL) for 5, 10 and 25 min. Fixed and permeabilized cells were stained with Anti-IKK total Recombinant Rabbit Polyclonal Antibody (Product # 710719, 1 µg/mL) or Anti-IKK alpha/beta (pS176/pS180) Recombinant Rabbit Polyclonal Antibody (Product # 710676, 1 µg/mL) or Anti-NF-kB Recombinant Rabbit Polyclonal Antibody (Product # 710048, 1 µg/mL) and labeled with Goat anti-Rabbit IgG (H+L) Superclonal Secondary Antibody, Alexa Fluor® 488 conjugate (Product # A27034, 0.4 µg/mL, 1:2500). Images showing cytoplasmic staining of IKK total and NF-kB (panel a & e; green) in untreated cells. No significant basal levels of phospho-IKK alpha/beta (panel i; green) were detected. Treatment with TNF-a led to a significant increase in the levels of IKK (panel b-d; green) and IKK alpha/beta (pS176/pS180) (panel j-l; green) in the cytosol, specifically at the perinucleus, and a corresponding translocation of NF-KB to the nucleus (panel f-h; green). No background staining was observed in control cells with no primary antibody (panel m).The nuclei (blue) were stained using SlowFade® Gold Antifade Mountant with DAPI (Product # S36938, 1:50). Cytoskeletal F-actin staining (red) was done using Alexa Fluor® 594 Phalloidin (Product # A12381, 1:200).

Supportive validation

Submitted by

Invitrogen Antibodies (provider)

Main image

Experimental details

Immunofluorescence was performed on HeLa cells treated with TNF alpha at 50 ng/mL/25 min. These cells were fixed and permeabilized for detection of IKK alpha/beta (pSpS 176/180) using Anti-IKK alpha/beta (pSpS 176/180) Recombinant Rabbit Polyclonal Antibody (Product # 710676, 1 µg/mL) and labeled with Goat anti-Rabbit IgG (H+L) Superclonal Secondary Antibody, Alexa Fluor® 488 conjugate (Product # A27034, 0.4 µg/mL, 1:2500). Panel a) shows representative cells that were stained for detection and localization protein (green), Panel b) is stained for nuclei (blue) using SlowFade® Gold Antifade Mountant with DAPI (Product # S36938, 1:50). Panel c) represents cytoskeletal F-actin staining using Alexa Fluor® 594 Phalloidin (Product # A12381, 1:200). Panel d) is a composite image of Panels a, b and c clearly demonstrating cytoplasmic and peri-nuclear localization of IKK alpha/beta (pSpS 176/180). Panel e represents control cells with no primary Antibody to assess background. Panel f) shows untreated cells.

Supportive validation

Submitted by

Invitrogen Antibodies (provider)

Main image

Experimental details

Immunofluorescence analysis of Phospho-IKK alpha/beta (Ser176, Ser180) was done on 70% confluent log phase ME-180 cells. The cells were either mock treated or treated with TNF-alpha (50 ng/mL for 20 min), fixed with 4% paraformaldehyde for 15 minutes, permeabilized with 0.1% Triton™ X-100 for 10 minutes, and blocked with 1% BSA for 1 hour at room temperature. The cells were subsequently labeled with Phospho-IKK alpha/beta (Ser176, Ser180 - Green) Recombinant Rabbit Polyclonal Antibody (Product # 710676) at 2µg/mL in 0.1% BSA and incubated for 3 hours at room temperature and then labeled with Goat anti-Rabbit IgG (H+L) Superclonal™ Secondary Antibody, Alexa Fluor® 488 conjugate (Product # A27034) at a dilution of 1:2000 for 45 minutes at room temperature. Nuclei (Blue) were stained with SlowFade® Gold Antifade Mountant DAPI (Product # S36938). F-actin (Red) was stained with Rhodamine Phalloidin (Product # R415, 1:300). TNFalpha induced phosphorylation of IKK alpha/beta, was observed in control cell line (panels a, c) and not in the IKK alpha knockout (KO) cell line (panels b, d). The images were captured at 40X magnification.