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: Establishment and titration of the optimal form of the substrate PIP 2 . A , the optimal form of substrate was determined by comparing two different ratios of lPIP 2 to total lipids, 1:10 and 1:2, both at a final lPIP 2 concentration of 7 μM. A water-soluble PIP 2 was also tested, at a concentration of 100 μM. Note that the most advantageous substrate preparation is a 1:2 mixture of lPIP 2 to total lipids. Two replicates from n = 10. B , different concentrations of lPIP 2 (1:2 mixture of lPIP 2 to total lipids) were tested. Note the dose-dependent effect. Two replicates from n = 4. C , schematic diagram of the experimental set up used in ( A ) and ( B ). 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. Specific activity values for PI3Kα, calculated as pmol PIP 3 produced per minute per nanogram of enzyme, are indicated in red fonts in the figure. lPIP 2 , lipid form of PIP 2 ; PIP 2 , phosphatidylinositol-4,5-biphospate; PIP 3 , phosphatidylinositol-3,4,5-triphosphate.

Article Snippet: Lipid PIP 2 [L-α-phosphatidylinositol-4,5 bisphosphate (Brain, Porcine) (ammonium salt)] and soluble PIP 2 [1,2-dioctanoyl-sn-glycero-3-phospho-(1′-myo-inositol-4′,5-bisphosphate) (ammonium salt)] were from Avanti Polar Lipids.

Techniques: Titration, Concentration Assay, Activity Assay, Produced

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: Role of membranes in the overactivation of the mutants H1047R and E545K. We compare the PI3Kα enzymatic activity of H1047R, E545K, and WT, using as substrate 7 μM lPIP 2 ( A ), or 100 μM sPIP 2 ( B ). The activities of the mutants were normalized to WT, which was set to value “1.” Note that E545K shows higher activity than WT with either soluble or membranous PIP 2 , whereas H1047R exhibits increased activity only when membranes are used. Two replicates from n = 6 in ( A ) and n = 7 in ( B ). The individual data points displayed above represent the means of replicates in each experiment. The statistical significance of difference between groups in graphs ( A ) and ( B ) was examined by one-way ANOVA, followed by Tukey’s multiple comparisons test. ns : no significance, ∗∗ p < 0.01, ∗∗∗ p < 0.001, and ∗∗∗∗ p < 0.0001. Data were shown as mean ± SD. Specific activity values for PI3Kα, calculated as pmol PIP 3 produced per minute per nanogram of enzyme, are indicated in red fonts in the figure. lPIP 2 , lipid form of phosphatidylinositol-4,5-biphospate; SPR, surface plasmon resonance; sPIP 2 , soluble form of PIP 2 ; PIP 3 , phosphatidylinositol-3,4,5-triphosphate.

Article Snippet: Lipid PIP 2 [L-α-phosphatidylinositol-4,5 bisphosphate (Brain, Porcine) (ammonium salt)] and soluble PIP 2 [1,2-dioctanoyl-sn-glycero-3-phospho-(1′-myo-inositol-4′,5-bisphosphate) (ammonium salt)] were from Avanti Polar Lipids.

Techniques: Activity Assay, Produced, SPR Assay