GSK3 is a key regulator in several physiological processes, such as cell cycle, oncogenesis and apoptosis in neuronal and vascular smooth muscle cells. Many of the pathways that use GSK3 as a regulator have links to human diseases.
GSK3-alpha is highly phosphorylated at Tyr279 in the phosphorylation loop. Constitutive phosphorylation of this Tyrosine is important for kinase activity. Dephosphorylation of Tyr279 after mitogen activation is accompanied by kinase inactivation. PKA as well as PI3K-activated PKB inactivate GSK3-alpha by phosphorylation at ser21.

As immunoprecipitation of GSK3 is a critical step to analysis of GSK3 kinase activity, we directly conjugated a high affinity monoclonal anti-GSK3-alpha antibody to Sepharose 4B beads to allow rapid and specific immoprecipitation of GSK3 without secondary reagents.

With another HRP-conjugated anti-GSK3-alpha antibody, the immunoprecipitated GSK3? can be directly detected by Western blot analysis.





Two-color, 4-plex western blot analysis of GSK3. MDA321 tumor cell cells were treated with lithium (lane 2 and 4) overnight, or remained untreated (lane 1 and 3). Lithium treatment causes GSK phosphorylation. Total cell lysates were separated in 10% SDS-PAGE and blotted onto nitrocellulose membrane. The blot was probed with four-antibody cocktail containing anti-total GSK3-alpha (mIgG1), anti-total GSK3-alpha (mIgG2a), rabbit anti-p-GSK3-alpha and anti-p-GSK3-alpha. After wash, the blot was further probed with a secondary antibody cocktail containing Cy5-conjugated anti-mIgG1 and IgG2a (red color) and FITC anti-rabbit IgG and anti-mIgG2b (green color). The image was revealed by Bio-Rad three-laser imager FX. A. image from Cy5 channel, B. Image from FITC channel. C. Two-color merged image.

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