Journal: eLife

Article Title: A peptide-neurotensin conjugate that crosses the blood-brain barrier induces pharmacological hypothermia associated with anticonvulsant, neuroprotective, and anti-inflammatory properties following status epilepticus in mice

doi: 10.7554/eLife.100527

Figure Lengend Snippet: ( A, C, and E ) Chemical structure and molecular weight of the VH-N21, VH-N41, and VH-N412 conjugates, containing the eight amino acid cyclic brain penetrating peptide that recognizes the LDLR (VH445 for VH-N21 and VH4129 for VH-N41 and VH-N412), and either the neurotensin (NT) tridecapeptide (VH-N21 and VH-N41) or its C-terminal NT(8–13) fragment (VH-N412). ( B, D, and F ) Hypothermic response to VH-N21, VH-N41, and VH-N412 conjugates in mice after single i.v. (bolus) injection at increasing dose levels. Core body (rectal) temperature was measured before (baseline) and at indicated times after injection. Data are presented as means ± SEM, n=4–8 per group. ( G ) Dose-response curves of VH-N21, VH-N41, and VH-N412 hypothermic response. ED 50 values for each conjugate were estimated by plotting the response vs log[dose(mg/kg eq. NT)] followed by nonlinear regression (three parameters) using GraphPad Prism software.

Article Snippet: Recombinant human LDLR (His-tagged) was purchased from Sino Biological (Beijing, China).

Techniques: Molecular Weight, Injection, Software

Journal: eLife

Article Title: A peptide-neurotensin conjugate that crosses the blood-brain barrier induces pharmacological hypothermia associated with anticonvulsant, neuroprotective, and anti-inflammatory properties following status epilepticus in mice

doi: 10.7554/eLife.100527

Figure Lengend Snippet: ( A ) Surface plasmon resonance (SPR) sensorgrams of the free VH4129 and the VH-N412 compound on immobilized human LDLR (hLDLR). Red lines show the specific binding of molecules obtained after double subtraction of the signal measured on the control flow cell (without immobilized LDLR) and a blank run. Black lines show fit curves of the experimental data with a 1:1 binding model. The illustrated data are representative of two to five independent experiments. ( B ) Dose-response inhibition curves of tritiated NT, bound on hNTSR-1 or rNTSR-1 membrane extracts, in the presence of indicated concentrations of NT or VH-N412. Indicated Ki values were estimated from mean IC50 values obtained by logarithmic regression of experimental data. Data were plotted as means ± SD of biological duplicates. ( C and D ) Comparison of degradation rates for NT or peptide-NT conjugates in mouse blood. NT or peptide-NT conjugates were incubated in freshly collected mouse ( C ) or human ( D ) blood and analyzed using liquid chromatography-tandem mass spectrometry (LC-MS/MS) at indicated times in the plasma fraction. Data were plotted as means ± SD of n=3 biological replicates. T 1/2 values were estimated from nonlinear regression (one-phase decay) of experimental data. ( E ) Blood-brain barrier (BBB) transport of tritium-labeled NT or peptide-NT conjugates using in situ brain perfusion in mice. Data were presented as mean ± SEM for three to six animals. Student’s t-test vs NT: *p<0.05, **p<0.01.

Article Snippet: Recombinant human LDLR (His-tagged) was purchased from Sino Biological (Beijing, China).

Techniques: SPR Assay, Binding Assay, Control, Inhibition, Membrane, Comparison, Incubation, Liquid Chromatography, Mass Spectrometry, Liquid Chromatography with Mass Spectroscopy, Labeling, In Situ

Journal: eLife

Article Title: A peptide-neurotensin conjugate that crosses the blood-brain barrier induces pharmacological hypothermia associated with anticonvulsant, neuroprotective, and anti-inflammatory properties following status epilepticus in mice

doi: 10.7554/eLife.100527

Figure Lengend Snippet:

Article Snippet: Recombinant human LDLR (His-tagged) was purchased from Sino Biological (Beijing, China).

Techniques: Transfection, Construct, Plasmid Preparation, Membrane, Binding Assay, Sequencing, RNA Binding Assay, Recombinant, DC Protein Assay, Software

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: In cellular experiments, human LDLR ECD purchased from R&D Systems (2148-LD/CF-MTO) was used interchangeably with internally generated materials.

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: In cellular experiments, human LDLR ECD purchased from R&D Systems (2148-LD/CF-MTO) was used interchangeably with internally generated materials.

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: In cellular experiments, human LDLR ECD purchased from R&D Systems (2148-LD/CF-MTO) was used interchangeably with internally generated materials.

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

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: To test how uptake of lipApoE is mediated by cell surface receptors, pHrodo green-lipApoE was pre-incubated with LDLR ECD (R&D Systems #2148-LD/CF-MTO, or generated as described above), low-molecular-weight heparin (Fisher Scientific, BP2524), or CD98hc ECD84 at 37 C for 30–40 min before being added to cells.

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: To test how uptake of lipApoE is mediated by cell surface receptors, pHrodo green-lipApoE was pre-incubated with LDLR ECD (R&D Systems #2148-LD/CF-MTO, or generated as described above), low-molecular-weight heparin (Fisher Scientific, BP2524), or CD98hc ECD84 at 37 C for 30–40 min before being added to cells.

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: To test how uptake of lipApoE is mediated by cell surface receptors, pHrodo green-lipApoE was pre-incubated with LDLR ECD (R&D Systems #2148-LD/CF-MTO, or generated as described above), low-molecular-weight heparin (Fisher Scientific, BP2524), or CD98hc ECD84 at 37 C for 30–40 min before being added to cells.

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

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: REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies Goat anti-human LDLR R&D Systems Cat: AF2148; RRID:AB_2135126 Rabbit anti-human ApoE Abcam Cat: ab52607; RRID:AB_867704 Mouse pan anti-human ApoE Cell Signaling Cat: 74417; RRID:AB_3094529 Mouse anti-LAMP1 DSHB Clone H4A3; RRID:AB_2296838 Rabbit anti-PLIN2 (ADFP) ThermoFisher Scientific Cat: MA5-32664; RRID:AB_2809941 Mouse anti-TuJ1 (b3-tubulin) Cell Signaling Cat: 4466S; RRID:AB_1904176 Chicken anti-MAP2 (microtubule-associated protein 2) Abcam Cat: ab5392; RRID:AB_2138153 Mouse anti-4-HNE Abcam Cat: ab48506; RRID:AB_867452 AT8 (Mouse anti-pS202/T205 tau) ThermoFisher Scientific Cat: MN1020; RRID:AB_223647 Goat anti-Iba1 Novus Cat: NB100-1028; RRID:AB_3148646 or AB_521594 Rabbit anti-NeuN Abcam Cat: ab177487; RRID:AB_2532109 Donkey anti-mouse 488nm ThermoFisher Scientific Cat: A-21202 Donkey anti-rabbit 488nm ThermoFisher Scientific Cat: A-21206 Donkey anti-rabbit 488nm PLUS ThermoFisher Scientific Cat: A-32790 Donkey anti-rabbit 568nm ThermoFisher Scientific Cat: A-10042 Donkey anti-mouse 647 nm ThermoFisher Scientific Cat: A-31571 Donkey anti-rabbit 647nm ThermoFisher Scientific Cat: A-31573 Donkey anti-goat 647nm ThermoFisher Scientific Cat: A-21447 Donkey anti-chicken 647nm ThermoFisher Scientific Cat: A-78952 Biological samples Human CSF samples Precision Med 7005, 8009, and 8200 Chemicals, peptides, and recombinant proteins ApoE2, ApoE3, R136S ApoE3, ApoE4, R136S ApoE4, K146E ApoE4 Generated in-house N/A LDLR ECD R&D Systems; or generated in-house Cat: 2148-LD/CF-MTO Heparin Fisher Scientific Cat: BP2524 DiI-LDL ThermoFisher Scientific Cat: L3482 HDL EMD Millipore Cat: LP3-5MG BODIPY-CE ThermoFisher Scientific Cat: C3927MP CE(18:1) Avanti Polar Lipids Cat: 700269 CE(20:4) Cayman Chem Cat: 22595 POPC Avanti Polar Lipids Cat: 850457 PBS Gibco Cat: 10010023 Bio-Beads SM-2 Resin Bio-Rad Cat: 1523920 Lipofectamine LTX-Plus Invitrogen Cat: A12621 Bovine Serum Albumin Sigma-Aldrich Cat: A2153-500G NucBlue Live ReadyProbes ThermoFisher Scientific Cat: R37605 DAPI Invitrogen Cat: D1306 Fluorescein-transferrin ThermoFisher Scientific Cat: T2871 LysoTracker Deep Red Invitrogen Cat: L12492 InstantBlue Abcam Cat: ab119211 ProLong Gold Antifade Mountant ThermoFisher Scientific Cat: P36930 (Continued on next page) ll OPEN ACCESS Cell 188, 1–20.e1–e13, January 9, 2025 e1 Please cite this article in press as: Guo et al., Decreased lipidated ApoE-receptor interactions confer protection against pathogenicity of ApoE and its lipid cargoes in lysosomes, Cell (2025), https://doi.org/10.1016/j.cell.2024.10.027 Article

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: REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies Goat anti-human LDLR R&D Systems Cat: AF2148; RRID:AB_2135126 Rabbit anti-human ApoE Abcam Cat: ab52607; RRID:AB_867704 Mouse pan anti-human ApoE Cell Signaling Cat: 74417; RRID:AB_3094529 Mouse anti-LAMP1 DSHB Clone H4A3; RRID:AB_2296838 Rabbit anti-PLIN2 (ADFP) ThermoFisher Scientific Cat: MA5-32664; RRID:AB_2809941 Mouse anti-TuJ1 (b3-tubulin) Cell Signaling Cat: 4466S; RRID:AB_1904176 Chicken anti-MAP2 (microtubule-associated protein 2) Abcam Cat: ab5392; RRID:AB_2138153 Mouse anti-4-HNE Abcam Cat: ab48506; RRID:AB_867452 AT8 (Mouse anti-pS202/T205 tau) ThermoFisher Scientific Cat: MN1020; RRID:AB_223647 Goat anti-Iba1 Novus Cat: NB100-1028; RRID:AB_3148646 or AB_521594 Rabbit anti-NeuN Abcam Cat: ab177487; RRID:AB_2532109 Donkey anti-mouse 488nm ThermoFisher Scientific Cat: A-21202 Donkey anti-rabbit 488nm ThermoFisher Scientific Cat: A-21206 Donkey anti-rabbit 488nm PLUS ThermoFisher Scientific Cat: A-32790 Donkey anti-rabbit 568nm ThermoFisher Scientific Cat: A-10042 Donkey anti-mouse 647 nm ThermoFisher Scientific Cat: A-31571 Donkey anti-rabbit 647nm ThermoFisher Scientific Cat: A-31573 Donkey anti-goat 647nm ThermoFisher Scientific Cat: A-21447 Donkey anti-chicken 647nm ThermoFisher Scientific Cat: A-78952 Biological samples Human CSF samples Precision Med 7005, 8009, and 8200 Chemicals, peptides, and recombinant proteins ApoE2, ApoE3, R136S ApoE3, ApoE4, R136S ApoE4, K146E ApoE4 Generated in-house N/A LDLR ECD R&D Systems; or generated in-house Cat: 2148-LD/CF-MTO Heparin Fisher Scientific Cat: BP2524 DiI-LDL ThermoFisher Scientific Cat: L3482 HDL EMD Millipore Cat: LP3-5MG BODIPY-CE ThermoFisher Scientific Cat: C3927MP CE(18:1) Avanti Polar Lipids Cat: 700269 CE(20:4) Cayman Chem Cat: 22595 POPC Avanti Polar Lipids Cat: 850457 PBS Gibco Cat: 10010023 Bio-Beads SM-2 Resin Bio-Rad Cat: 1523920 Lipofectamine LTX-Plus Invitrogen Cat: A12621 Bovine Serum Albumin Sigma-Aldrich Cat: A2153-500G NucBlue Live ReadyProbes ThermoFisher Scientific Cat: R37605 DAPI Invitrogen Cat: D1306 Fluorescein-transferrin ThermoFisher Scientific Cat: T2871 LysoTracker Deep Red Invitrogen Cat: L12492 InstantBlue Abcam Cat: ab119211 ProLong Gold Antifade Mountant ThermoFisher Scientific Cat: P36930 (Continued on next page) ll OPEN ACCESS Cell 188, 1–20.e1–e13, January 9, 2025 e1 Please cite this article in press as: Guo et al., Decreased lipidated ApoE-receptor interactions confer protection against pathogenicity of ApoE and its lipid cargoes in lysosomes, Cell (2025), https://doi.org/10.1016/j.cell.2024.10.027 Article

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: REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies Goat anti-human LDLR R&D Systems Cat: AF2148; RRID:AB_2135126 Rabbit anti-human ApoE Abcam Cat: ab52607; RRID:AB_867704 Mouse pan anti-human ApoE Cell Signaling Cat: 74417; RRID:AB_3094529 Mouse anti-LAMP1 DSHB Clone H4A3; RRID:AB_2296838 Rabbit anti-PLIN2 (ADFP) ThermoFisher Scientific Cat: MA5-32664; RRID:AB_2809941 Mouse anti-TuJ1 (b3-tubulin) Cell Signaling Cat: 4466S; RRID:AB_1904176 Chicken anti-MAP2 (microtubule-associated protein 2) Abcam Cat: ab5392; RRID:AB_2138153 Mouse anti-4-HNE Abcam Cat: ab48506; RRID:AB_867452 AT8 (Mouse anti-pS202/T205 tau) ThermoFisher Scientific Cat: MN1020; RRID:AB_223647 Goat anti-Iba1 Novus Cat: NB100-1028; RRID:AB_3148646 or AB_521594 Rabbit anti-NeuN Abcam Cat: ab177487; RRID:AB_2532109 Donkey anti-mouse 488nm ThermoFisher Scientific Cat: A-21202 Donkey anti-rabbit 488nm ThermoFisher Scientific Cat: A-21206 Donkey anti-rabbit 488nm PLUS ThermoFisher Scientific Cat: A-32790 Donkey anti-rabbit 568nm ThermoFisher Scientific Cat: A-10042 Donkey anti-mouse 647 nm ThermoFisher Scientific Cat: A-31571 Donkey anti-rabbit 647nm ThermoFisher Scientific Cat: A-31573 Donkey anti-goat 647nm ThermoFisher Scientific Cat: A-21447 Donkey anti-chicken 647nm ThermoFisher Scientific Cat: A-78952 Biological samples Human CSF samples Precision Med 7005, 8009, and 8200 Chemicals, peptides, and recombinant proteins ApoE2, ApoE3, R136S ApoE3, ApoE4, R136S ApoE4, K146E ApoE4 Generated in-house N/A LDLR ECD R&D Systems; or generated in-house Cat: 2148-LD/CF-MTO Heparin Fisher Scientific Cat: BP2524 DiI-LDL ThermoFisher Scientific Cat: L3482 HDL EMD Millipore Cat: LP3-5MG BODIPY-CE ThermoFisher Scientific Cat: C3927MP CE(18:1) Avanti Polar Lipids Cat: 700269 CE(20:4) Cayman Chem Cat: 22595 POPC Avanti Polar Lipids Cat: 850457 PBS Gibco Cat: 10010023 Bio-Beads SM-2 Resin Bio-Rad Cat: 1523920 Lipofectamine LTX-Plus Invitrogen Cat: A12621 Bovine Serum Albumin Sigma-Aldrich Cat: A2153-500G NucBlue Live ReadyProbes ThermoFisher Scientific Cat: R37605 DAPI Invitrogen Cat: D1306 Fluorescein-transferrin ThermoFisher Scientific Cat: T2871 LysoTracker Deep Red Invitrogen Cat: L12492 InstantBlue Abcam Cat: ab119211 ProLong Gold Antifade Mountant ThermoFisher Scientific Cat: P36930 (Continued on next page) ll OPEN ACCESS Cell 188, 1–20.e1–e13, January 9, 2025 e1 Please cite this article in press as: Guo et al., Decreased lipidated ApoE-receptor interactions confer protection against pathogenicity of ApoE and its lipid cargoes in lysosomes, Cell (2025), https://doi.org/10.1016/j.cell.2024.10.027 Article

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