Journal: Frontiers in Molecular Neuroscience
Article Title: Novel Non-phosphorylated Serine 9/21 GSK3β/α Antibodies: Expanding the Tools for Studying GSK3 Regulation
Figure Lengend Snippet: Detection of recombinant npS9 GSK3β with 12B2 and 15C2 antibodies is linear and correlates with kinase activity. (A) The level of GSK3β kinase activity with 30, 60, 120, 180, 240, and 300 ng of npS9 GSK3β (“active”) was measured using an in vitro GSK3β kinase activity assay and there was a linear increase in kinase activity with increasing amounts of GSK3β ( r 2 = 0.93). Three independent experiments were performed. (B) For western blotting, recombinant GSK3β was incubated with alkaline phosphatase to generate nonphosphoS9 GSK3β or incubated with Akt1 to generate phosphoS9 GSK3β, and then 0, 30, 60, 120, 180, 240, or 300 ng of npS9 GSK3β was mixed with 300, 240, 180, 120, 60, or 0 ng of pS9 GSK3β to bring the total protein content to 300 ng/lane. The blot was probed with 12B2 (red) and total GSK3α/β antibodies (green). (C) Quantitation of signal from 12B2 shows a linear increase in reactivity with increasing npS9 GSK3β amount ( r 2 = 0.92). (D) The same samples were probed with 15C2 (red) and total GSK3α/β antibodies (green). (E) Quantitation of signal from 15C2 shows a linear increase in reactivity with increasing npS9 GSK3β amount ( r 2 = 0.90). It is notable that both 12B2 and 15C2 signals also showed a direct correlation with GSK3β activity levels (12B2: r = 0.99, p = 0.0002; 15C2: r = 0.99, p
Article Snippet: After sample incubation, the wells were rinsed and incubated in 50 μl/well GSK3β activity reaction buffer [100 μM ATP (V915A, Promega), 40 mM Tris-HCl, pH 7.4, 20 mM MgCl2 and 0.1 mg/ml bovine serum albumin] for 1 h at 30°C.
Techniques: Recombinant, Activity Assay, In Vitro, Kinase Assay, Western Blot, Incubation, Quantitation Assay