Supplementary Figure 4: Membrane binding of Notch1 juxtamembrane polybasic region.
From: Dynamic regulation of CD28 conformation and signaling by charged lipids and ions
(a) Sequence information and charge property of human Notch1 juxtamembrane domain.
(b) Incubation of 2 μM Notch1_JM with 0.1 mM acidic POPG LUVs led to significant increase of AFE value but such an increase was not observed in that with 0.1 mM zwitterionic POPC LUVs.
(c-d) Titration of acidic POPG LUVs with the indicated concentrations into 2 μM Notch1_JM sample led to the gradient increase of AFE value (at approximate 350nm). Three independent samples were measured for each condition. Unpaired t-test was used for the statistical analysis of each two groups. One-Way ANOVA was used to test whether POPG treatment could cause significant change of the AFE value, P < 0.0001.
(e-f) The de-quenching FRET was used to measure the interaction of Notch1 juxtamembrane polybasic region with the plasma membrane in live Jurkat T cells. Representative cells are shown in e. Data in f are representative of three independent experiments. Bars in e represent 2 μm. N = 31, 32, 35 for the three conditions (from left to right). Each dot represents the FRET value from one individual cell. One-Way ANOVA was used to analyze difference among three groups, the p-values of these three panels from left to right were <0.0001, 0.2074 and 0.1555. Unpaired t-test was used to analyze difference between each two groups.
The center value and error bar in d,f denote mean and s.e.m.. Source data for b-d, and f are available in Source Data 6.
