Anti NFkB p65 (RelA), phospho Ser276  

Rockland's anti-p65 pS276 antibody was diluted 1:500 to detect p65 in human kidney tissue. Tissue was formalin fixed and paraffin embedded. No pre-treatment of sample was required. The image shows the localization of antibody as the precipitated red signal, with a hematoxylin purple nuclear counter stain.

Anti-pS276 shows phospho p65 staining in carcinoma cells. Western blot of total protein lysates from various human head and neck tumors shows phospho p65 staining in tumor cell lines using phospho specific polyclonal anti-human pS276 p65. Lanes 1-6 contain protein lysates from human squamal carcinoma cell lines. Lane 7 is a protein lysate from a primary culture of human keratinocytes and does not show significant levels of phosphorylated p65. Lane 8 contains protein lysate from ATCC SCC9 cells (also a head and neck squamal carcinoma). Lane 9 contains lysate from EGF-induced human derived A431 cells. Lane 10 (not shown) contains a molecular weight standard. Concurrent staining with anti-beta microtubulin (not shown) was used to confirm equal protein loading in all lanes. HRP conjugated Gt-anti-Rabbit IgG was used to develop the blot using a chemiluminescent detection method. Other detection methods will yield similar results. Data contributed by Yu, M., NIH, personal communication.

TNF Induces phosphorylation of p65 in KBM-5 cells. Cytoplasmic and nuclear protein lysates prepared after 0, 5, 10, 15, 30 and 60 minutes of 0.1 nM TNF treatment of KBM-5 cells shows inducible phosphorylation using phospho specific polyclonal anti-human pS276 p65. Rockland Immunochemical's pan reactive anti p65 (code# 100-4165) was used a control to show the presence of total p65 in both the cytoplasmic and nuclear extracts. Phosphorylation of p65 occurs after approximately 10 min of TNF exposure. Migration of phosphorylated p65 into the nucleus occurs within a similar time frame. HRP conjugated Gt-anti-Rabbit IgG was used to develop the blot using a chemi -luminescent detection method. Other detection methods will yield similar results. Personal Communication, Aggarwal BB