The GSK3B pY216 ELISA kit was used to measure the levels of phosphorylated GSK3B in human (MCF7), mouse (MHH), rat (H4IIE) and monkey (COS7) sample lysates
Samples were loaded on 4-12% acrylamide gels as follows: Blot A = (1) 50 ng of GSK3B recombinant protein ab63193, Blot B and C = (2) NIH3T3 control, (3) NIH3T3 Mock treated, (4) NIH3T3 CIP treated and (5) Mouse Heart homogenate. Blots A and B were incubated overnight with the capture anti-GSK3B antibody whereas Blot C was incubated overnight with the detector anti-GSK3A+B (pY279+Y216). The capture shows targeting of the recombinant protein and a single band with crude extracts, showing the specificity of the antibody for the B isoform. The detector shows complete dephosphorylation of GSK3A and B after treatment with CIP (4) in comparison to the mock (3) and untreated (2) controls.
The standard curve was generated using Mouse Heart Homogenate (MHH).