I2PP2A/SET Detection Kit

Figure: Nuclear localization of I2PP2A/SET using Anti I2PP2A/SET. Confluent HEK293 Cells were coated with poly-D-lysine and fixed onto sterile glass cover slips with methanol. The cells were permeablized with acetone and incubated with bovine serum albumin. The albumin was removed and the cells were incubated with human Lamin B Monoclonal antibody 101-B7 (panel A) or Anti-I2PP2A/SET at 1:100 dilution (panel B). After washing with PBS, cells were incubated with goat anti-mouse IgG (H+L) absorbed against rabbit serum and conjugated to Lissamine Rhodamine (panel A) or goat anti-rabbit IgG (H+L) absorbed against mouse serum and conjugated to Cy2. Cover slips were washed three times with PBS, mounted onto glass slides, and viewed at 20 x magnification using a fluorescence microscope. Panels A and B show the cells stained with Lissamine Rhodamine and Cy2, respectively. Panel C shows Panels A and B superimposed. Control cells probed with the secondary antibody alone or with preimmune serum and the secondary antibody displayed no reaction (not shown).

Background: I2PP2A/SET is  a potent inhibitor of Protein Phosphatase 2A (PP2A), a major mammalian protein serine/threonine phosphatase that regulates diverse cellular processes. Purified preparations of I2PP2A/SET inhibit all forms of the phosphatase tested to date in a substrate selective manner. For example, the preparations inhibit PP2A potently (ki ~ 0.1 – 0.5 nM) with Myelin Basic Protein (MBP) and Histone H1 but not with Casein as substrate.  I2PP2A/SET was found to occur as a fusion protein with the nucleoporin CAN (NUP214) in a patient with acute undifferentiated leukemia. Elevated expression of I2PP2A/SET has been shown to occur in a number of other cancers.  Transient expression of I2PP2A/SET in HEK-293 cells leads to an increase in the phosphorylation of c-Jun at Ser63 and Ser73, and DNA binding and AP1 activities of the proto-oncogene. The sphingolipid second messenger cermaide binds to I2PP2A/SET and prevents it from inhibiting PP2A. This may contribute, at least in part, to the mechanism by which Ceramide promotes apoptosis. I2PP2A/SET also prevents inhibition of E-CDK-2 by p21Cip1, associates with B cyclin, regulates histone binding to DNA, transcriptional activity and chromatin condensation. I2PP2A/SET undergoes phosphorylation at Ser9 and Ser24 in intact cells. The functional significance of these phosphorylations is uncertain although some studies indicate that Ser9 phosphorylation may causes cytoplasmic detention of I2PP2A/SET in Alzheimer disease.

References: Li M, Makkinje A, Damuni Z.  (1996)  J Biol Chem 271, 11059-11062; von Lindern, M. et al (1992)  Mol Cell Biol 12, 3346-3355; Al-Murrani, S. W., Woodgett, J. R., and Damuni, Z. (1999) Biochem J. 341, 293–298; Li, M., Damuni, Z. (1998) Proteins” Methods Mol Biol  93, 59-66; Katayose, Y. et al (2000)  J Biol Chem 275, 9209-9214