Journal: The Journal of Biological Chemistry
Article Title: A new functional assay reveals that membrane binding is critical for overactivation of the phosphoinositide 3-kinase H1047R mutant
doi: 10.1016/j.jbc.2026.111207
Figure Lengend Snippet: Principle of the PI3Kα activity assay and titration of its basic components GST-GRP1 and b-PIP 3 . A , table listing the key steps (1–5) of the assay. B , schematic illustration of the assay’s principle. The number of each individual step of the assay in ( B ) corresponds to the same number of the step shown in ( A ) (numbers are shown in red fonts , both in ( A ) and ( B ). In step 1, GST-GRP1 is immobilized to the walls of the plate. In step 2, in the absence of PI3Kα (negative control), the plates are incubated with b-PIP 3 alone. In the presence of PI3Kα, the enzymatic reactions are set up in separate tubes (step 2a), b-PIP 3 is added to the tubes post termination of the PI3Kα reaction (step 2b) and the mixture is added to the wells (step 2c), where PIP 3 (generated by PI3Kα) competes with b-PIP 3 (added exogenously) for binding to GST-GRP1. Next, streptavidin-HRP is added (step 3) to assess the amount of bound b-PIP 3 , followed by the addition of OPD (step 4), the specific substrate of HRP, used to generate a chromophore measured at 492 nm (step 5). C , the optimal amount of GST-GRP1 in the assay was determined by testing increasing concentrations of this component, followed by addition of 0.9 ng b-PIP 3 . Two replicates from n = 5. D , the optimal amount of b-PIP 3 was assessed by adding varying quantities of b-PIP 3 to glutathione-coated wells, preincubated with 4 μg GST-GRP1. Two replicates from n = 5. The individual data points displayed above represent the means of replicates in each experiment. The statistical significance of difference between groups was examined by one-way ANOVA, followed by Tukey’s multiple comparisons test. ns , no significance, ∗∗∗ p < 0.001, and ∗∗∗∗ p < 0.0001. Data were shown as mean ± SD. GRP, general receptor for phosphoinositide; GST, glutathione-S-transferase; PIP 3 , phosphatidylinositol-3,4,5-triphosphate; b-PIP 3 , biotin-phosphatidylinositol-3,4,5-triphosphate; HRP, horseradish peroxidase.
Article Snippet: Glutathione-coated plates as well as streptavidin poly-HRP were from PIERCE/Thermo Fisher Scientific.
Techniques: Activity Assay, Titration, Negative Control, Incubation, Generated, Binding Assay
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