Confocal immunofluorescent analysis of MEF/GSK-3 wildtype cells (left), MEF/GSK-3β (-/-) cells (center) and MEF/GSK-3α (-/-) cells (right), using GSK-3α (D80D1) XP® Rabbit mAb (green). Actin filaments have been labeled with DY-554 phalloidin (red). Blue pseudocolor = DRAQ5® #4084 (fluorescent DNA dye). (MEF/wildtype, GSK-3α (-/-) and GSK-3β (-/-) cells were kindly provided by Dr. Jim Woodgett, University of Toronto, Canada).
Supplied in 10 mM sodium HEPES (pH 7.5), 150 mM NaCl, 100 µg/ml BSA, 50% glycerol and less than 0.02% sodium azide. Store at –20°C. Do not aliquot the antibody.
Achieve higher quality immunofluorescent images using the efficient and cost-effective, pre-made reagents in our #12727 Immunofluorescence Application Solutions Kit
NOTE: Prepare solutions with reverse osmosis deionized (RODI) or equivalent grade water.
Recommended Fluorochrome-conjugated Anti-Rabbit secondary antibodies:
NOTE: Cells should be grown, treated, fixed and stained directly in multi-well plates, chamber slides or on coverslips.
Aspirate liquid, then cover cells to a depth of 2–3 mm with 4% formaldehyde diluted in 1X PBS.
NOTE: Formaldehyde is toxic, use only in a fume hood.
NOTE: All subsequent incubations should be carried out at room temperature unless otherwise noted in a humid light-tight box or covered dish/plate to prevent drying and fluorochrome fading.
posted November 2006
revised November 2013
Protocol Id: 24
GSK-3α (D80D1) XP® Rabbit mAb detects endogenous levels of total GSK-3α protein. The antibody does not cross-react with GSK-3β.
Monoclonal antibody is produced by immunizing animals with a synthetic peptide corresponding to the sequence of human GSK-3α.
Glycogen synthase kinase-3 (GSK-3) was initially identified as an enzyme that regulates glycogen synthesis in response to insulin (1). GSK-3 is a ubiquitously expressed serine/threonine protein kinase that phosphorylates and inactivates glycogen synthase. GSK-3 is a critical downstream element of the PI3K/Akt cell survival pathway whose activity can be inhibited by Akt-mediated phosphorylation at Ser21 of GSK-3α and Ser9 of GSK-3β (2,3). GSK-3 has been implicated in the regulation of cell fate in Dictyostelium and is a component of the Wnt signaling pathway required for Drosophila, Xenopus, and mammalian development (4). GSK-3 has been shown to regulate cyclin D1 proteolysis and subcellular localization (5).
GSK-3α regulates the production of amyloid-β peptides, a major component of the plaques that accumulate with progression of Alzheimer disease. Administration of therapeutic concentrations of lithium, a GSK-3 inhibitor, attenuates amyloid-β production by specifically inhibiting the cleavage of amyloid precursor protein (APP) by γ-secretase, blocking accumulation of amyloid-β peptides in the brains of mice that overproduce APP (6).
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