Application Brief 1005: GSK3-alpha NanoPro Assay

GSK3 is a critical downstream element of the PI3 kinase/AKT cell survival pathway whose activity can be inhibited by AKT-mediated phosphorylation at Ser21 of GSK-3α and Ser9 of GSK-3β. While GSK-3α and GSK-3β have high sequence homology, their biological function differs. The data presented shows an increase of peaks detected by anti-phospho GSK-3α antibody as well as an increase of the same peaks detected by the total anti-GSK-3α antibody in response to EGF treatment in MCF10A cells. At the same time, the peaks not recognized by the anti-phospho Ser21 antibody decrease in size, implying that these peaks represent non-phospho or non-pS21 phospho GSK-3α forms. The position of these peaks around pI 9 is in accordance with the theoretical pI for this sequence. In other cell systems (A549 for example), both the anti-total GSK-3 antibody as well as the anti-phospho GSK-3α antibody also recognize peaks around 6.0. Alignment of the GSKα and GSKβ profiles in addition to use of antibodies recognizing both isoforms confirmed the specificity of the anti-GSK-3α versus the anti-GSK-3β antibodies used (data not shown).