popc cholesterol  (Croda International Plc)

Journal: Cell

Article Title: Decreased lipidated ApoE-receptor interactions confer protection against pathogenicity of ApoE and its lipid cargoes in lysosomes.

doi: 10.1016/j.cell.2024.10.027

Figure Lengend Snippet: Figure 1. ApoE isoform-dependent LDLR binding results in differential cellular uptake (A and B) Confocal images and quantification of FITC-labeled POPC-lipidated ApoE (10 mg/mL) bound to surface of 293T cells overexpressing LDLR after 1 h incubation at 4C. Scale bar: 10 mm in (A). (C) HTRF showing POPC-lipidated ApoE binding to LDLR ECD (3 independent experiments). (D) SPR profiles for POPC-lipidated ApoE isoform binding to biotinylated LDLR ECD. Red: experimental data. Black: curve fit using a 1:1 kinetic binding model. Each curve represents binding at one concentration (0.5, 1, and 2 mM for lipApoE2; 0.0156, 0.03125, and 0.0625 mM for lipApoE3/E4).

Article Snippet: Cell-based LDLR binding assay POPC/cholesterol-lipidated ApoE was labeled using FluoReporter FITC protein labeling kit (Invitrogen, F6434) with excess dye removed by dialysis into PBS using Slide-A-Lyzer MINI dialysis device with 10kDa MWCO (ThermoFisher, #69570).

Techniques: Binding Assay, Labeling, Incubation, Concentration Assay

Journal: Cell

Article Title: Decreased lipidated ApoE-receptor interactions confer protection against pathogenicity of ApoE and its lipid cargoes in lysosomes.

doi: 10.1016/j.cell.2024.10.027

Figure Lengend Snippet: Figure 4. Differential lipid burden modulates inflammatory responses and transcription of microglia (A and B) Confocal images and quantification of BODIPY signals in APOE KO iMg after 1-day incubation with 10 mg/mL BODIPY-CE pre-complexed with 10 mg/mL HDL and 10 mg/mL ApoE, with and without co-treatment of 20 mg/mL LDLR ECD, in medium containing 100 ng/mL LPS. Scale bar: 20 mm for (A). (C) Relative expression of 5 genes quantified by qPCR for APOE KO iMg after live imaging shown in (A).

Article Snippet: Cell-based LDLR binding assay POPC/cholesterol-lipidated ApoE was labeled using FluoReporter FITC protein labeling kit (Invitrogen, F6434) with excess dye removed by dialysis into PBS using Slide-A-Lyzer MINI dialysis device with 10kDa MWCO (ThermoFisher, #69570).

Techniques: Incubation, Expressing, Imaging

Journal: Cell

Article Title: Decreased lipidated ApoE-receptor interactions confer protection against pathogenicity of ApoE and its lipid cargoes in lysosomes.

doi: 10.1016/j.cell.2024.10.027

Figure Lengend Snippet: Figure 7. Christchurch mutation reduces LDLR binding, lipid uptake, and lipofuscin (A) Competitive HTRF demonstrating inhibition of tagged lipApoE4-LDLR ECD binding in the presence of different concentrations of untagged lipidated ApoE variants (n = 3 independent experiments). No inhibitor: no addition of untagged lipidated ApoE. The first panel shows the complete dose response. The second and third panels show the % inhibition at 125 and 250 nM, respectively. (B and C) Confocal images and quantification of pHrodo green-labeled POPC-lipidated ApoE isoforms (10 mg/mL) in H4 cells after 1-day incubation. Scale bar: 10 mm in (B). (D and E) Confocal images and quantification of pHrodo signals detected in H4 cells after 1-day incubation with 10 mg/mL pHrodo green-labeled HDL pre- complexed with 10 mg/mL ApoE isoforms. Scale bar: 10 mm in (D). (F and G) Confocal images and quantification of lipofuscin in H4 cells after 3-day incubation with 20 mg/mL CE(20:4)/POPC-lipidated ApoE isoforms. Scale bar: 10 mm in (F). In all bar graphs, data shown are mean + SEM with each dot representing one independent experiment; **p < 0.01, ***p < 0.001, ****p < 0.0001. One-way ANOVA was performed with Holm-Sidak’s multiple comparisons. See also Figure S7.

Article Snippet: Cell-based LDLR binding assay POPC/cholesterol-lipidated ApoE was labeled using FluoReporter FITC protein labeling kit (Invitrogen, F6434) with excess dye removed by dialysis into PBS using Slide-A-Lyzer MINI dialysis device with 10kDa MWCO (ThermoFisher, #69570).

Techniques: Mutagenesis, Binding Assay, Inhibition, Labeling, Incubation