Journal: Biochemistry

Article Title: High-affinity binding of LDL receptor–related protein 1 to matrix metalloprotease 1 requires protease:inhibitor complex formation

doi: 10.1021/acs.biochem.0c00442

Figure Lengend Snippet: A. MMP-1/TIMP-1 complex formation abolished MMP-1 protease activity as assessed by fluorescent substrate assay. Each form of MMP-1 (20 nM) was incubated with 200 µM of quenched fluorescent substrate. Fluorescence was measured every 20 seconds for 2 hours. All measurements were controlled for by background subtraction and then the relative fluorescence intensity was averaged across all replicates (n = 3). proMMP-1 (orange), active MMP-1 (blue), and MMP-1/TIMP-1 complex (gray). Inset. SDS PAGE analysis of proMMP-1 under non-reducing conditions. Lane 1, Standards, Lane 2, proMMP-1. B. Purified LRP1 was immobilized on a CM5 sensor chip and 75 nM proMMP-1 was injected in the absence (blue lines) or presence (orange lines) of 3 mM EDTA. C. RAP (1 μM, green) was injected (first arrow) on an LRP1-coated CM5 sensor chip (green) followed by either a co-injection (second arrow) of 75 nM proMMP-1 and 1 μM RAP (orange), a co-injection of buffer and 1 μM RAP (gray), or another injection of 1 μM RAP (green). These traces were compared to an injection of 75 nM proMMP-1 (blue) in the absence of RAP. D-E. Purified LRP1 was immobilized on a CM5 sensor chip and active MMP-1 (D) or MMP-1/TIMP-1 complex (E) were injected in the absence (blue lines) or presence (orange lines) of 3 mM EDTA. The data shown is a representative experiment from three independent experiments that were performed.

Article Snippet: Recombinant human LRP1 cluster II, III, and IV Fc chimera proteins were obtained from Molecular Innovations.

Techniques: Activity Assay, Incubation, Fluorescence, SDS Page, Purification, Injection

Journal: Biochemistry

Article Title: High-affinity binding of LDL receptor–related protein 1 to matrix metalloprotease 1 requires protease:inhibitor complex formation

doi: 10.1021/acs.biochem.0c00442

Figure Lengend Snippet: SPR experiments examined binding of TIMP-1 (A), TIMP-2 (B), TIMP-3 (C), or TIMP-4 (D) to full length LRP1. Concentrations used were: 0.58, 1.17, 2.34, 4.68, 9.38, 18.75, 37.5, 75, and 150 nM for TIMP-1; 4.68, 9.38, 18.75, 37.5, 75, and 150 nM for TIMP-2; 0.29, 0.58, 1.17, 2.34, 4.68, 9.38, 18.75, 37.5, 75, and 150 nM for TIMP-3 and TIMP-4. At each concentration, the binding association curves were fit to a pseudo-first order process to determine Req. Req values were then plotted versus concentration, and the data fit to a binding isotherm using GraphPad Prism 8.0 software. To normalize the data from different experiments, Req/Rmax was plotted versus ligand concentrations, and the data plotted shows mean ± SEM (n=3).

Article Snippet: Recombinant human LRP1 cluster II, III, and IV Fc chimera proteins were obtained from Molecular Innovations.

Techniques: Binding Assay, Concentration Assay, Software

Journal: Biochemistry

Article Title: High-affinity binding of LDL receptor–related protein 1 to matrix metalloprotease 1 requires protease:inhibitor complex formation

doi: 10.1021/acs.biochem.0c00442

Figure Lengend Snippet: Equilibrium binding constants for the interaction of TIMP-1, TIMP-2, TIMP-3, and TIMP-4 with LRP1. Equilibrium binding constants were calculated from equilibrium SPR measurements, in which Req was determined by fitting the association data to a pseudo-first-order process. Req was then plotted versus concentration and analyzed by non-linear regression analysis to determine the K D using GraphPad 8.0 software.

Article Snippet: Recombinant human LRP1 cluster II, III, and IV Fc chimera proteins were obtained from Molecular Innovations.

Techniques: Binding Assay, Concentration Assay, Software

Journal: Biochemistry

Article Title: High-affinity binding of LDL receptor–related protein 1 to matrix metalloprotease 1 requires protease:inhibitor complex formation

doi: 10.1021/acs.biochem.0c00442

Figure Lengend Snippet: Lysine residues on proMMP-1 or TIMP-1 were alkylated (Alk MMP-1 or Alk TIMP-1) by incubation with a 50-fold molar excess of Sulfo-NHS-acetate at 4°C for 2 hours. The alkylated proteins were then dialyzed into HBS + 1 mM CaCl2. Alk or unmodified proMMP-1 was then activated and complexed with either unmodified TIMP-1 or Alk TIMP-1. The experiment was repeated 3 times. (A) Activity for each MMP-1 species or complex was determined by fluorescent substrate assay using either 20 nM of unmodified or 40 nM of alkylated species or complex. For each replicate, background hydrolysis of the substrate was subtracted and the rate determined from the slope of the linear regression of the data. (B-E) LRP1 was immobilized on a CM5 chip and 75 nM of unmodified (blue) or 100 nM alkylated (orange) MMP-1 or TIMP-1 species or complex were injected over the chip. The data shown is a representative experiment from three independent experiments that were performed.

Article Snippet: Recombinant human LRP1 cluster II, III, and IV Fc chimera proteins were obtained from Molecular Innovations.

Techniques: Incubation, Activity Assay, Injection

Journal: Biochemistry

Article Title: High-affinity binding of LDL receptor–related protein 1 to matrix metalloprotease 1 requires protease:inhibitor complex formation

doi: 10.1021/acs.biochem.0c00442

Figure Lengend Snippet: (A) Schematic of the bivalent binding model used to fit the data. In this model, the MMP-1 ligand contains two regions (a-b) that interact with two LDLa ligand binding repeats on LRP1 (AB). The first region on MMP-1 (a) docks into an LDLa repeat (A) to form the initial complex. Then, the second region on MMP-1 (b) docks into the remaining LDLa repeat (B) to form the bivalent complex. (B) Increasing concentrations (4.7, 9.4, 18.7, 37.5, 75, and 150 nM) of proMMP-1 were injected over the LRP1-coated surface. (C) Increasing concentrations (4.7, 9.4, 18.7, 37.5, 75, and 150 nM) of active MMP-1 were injected over the LRP1-coated surface. (D) Increasing concentrations (2.3, 4.7, 9.4, 18.7, 37.5, 75, and 150 nM) of MMP-1/TIMP-1 complex were injected over the LRP1-coated surface. Fits of the experimental data (black lines) to a bivalent binding model are shown as blue lines. The data shown is a representative experiment from three independent experiments that were performed.

Article Snippet: Recombinant human LRP1 cluster II, III, and IV Fc chimera proteins were obtained from Molecular Innovations.

Techniques: Binding Assay, Ligand Binding Assay, Injection

Journal: Biochemistry

Article Title: High-affinity binding of LDL receptor–related protein 1 to matrix metalloprotease 1 requires protease:inhibitor complex formation

doi: 10.1021/acs.biochem.0c00442

Figure Lengend Snippet: Kinetic and equilibrium constants for the binding of proMMP-1, proMMP-9, MMP-1/TIMP-1 complexes, and proMMP-9/TIMP-1 complexes to LRP1.

Article Snippet: Recombinant human LRP1 cluster II, III, and IV Fc chimera proteins were obtained from Molecular Innovations.

Techniques: Binding Assay

Journal: Biochemistry

Article Title: High-affinity binding of LDL receptor–related protein 1 to matrix metalloprotease 1 requires protease:inhibitor complex formation

doi: 10.1021/acs.biochem.0c00442

Figure Lengend Snippet: hAoSMCs were plated at 7.2 × 10⁴ cells per well in a 12-well plate and were incubated with 125I-labeled proMMP-1 (25 nM) for 24 hours at 37°C in the absence or presence of RAP (2.5 μM) or the LRP1-specific polyclonal antibody R2629 (300 mg/mL). Following incubation, the amount of proMMP-1 internalized (A) degraded (B) or located on the cell surface (C) was measured. All data are plotted as mean ± SEM (n=3). Statistical significance was determined by one-way ANOVA with post-hoc Tukey’s test (*p<0.05, **p<0.01, ***p<0.0001).

Article Snippet: Recombinant human LRP1 cluster II, III, and IV Fc chimera proteins were obtained from Molecular Innovations.

Techniques: Incubation, Labeling

Journal: Biochemistry

Article Title: High-affinity binding of LDL receptor–related protein 1 to matrix metalloprotease 1 requires protease:inhibitor complex formation

doi: 10.1021/acs.biochem.0c00442

Figure Lengend Snippet: Recombinant LRP1 fragments of either Cluster II, III, or IV were immobilized to three individual flow cells of a CM5 sensor chip using an amine-reactive coupling process. SPR experiments tested binding of proMMP-1 (A) or TIMP-1 (B) to Cluster II (orange), III (blue), and IV (gray) with increasing concentrations of ligand (proMMP-1: 0.29, 0.58, 1.17, 2.34, 4.68, 9.38, 18.75, 37.5, 75, and 150 nM; TIMP-1: 0.58, 1.17, 2.34, 4.68, 9.38, 18.75, 37.5, 75, 150, and 300 nM). At each concentration, the binding association curves were fit to a pseudo-first order process. Req was estimated as the maximum number of response units at equilibrium for each concentration. Shown are the plots of Req versus ligand concentration. Data is plotted as mean ± SEM (n=3). The data were normalized to the amount of cluster coated on the CM5 sensor chip. GraphPad Prism 8.0 software was used to fit the data to the specific binding non-linear regression model to determine the KD of each ligand for each cluster. All calculated KD values are reported in Table 3.

Article Snippet: Recombinant human LRP1 cluster II, III, and IV Fc chimera proteins were obtained from Molecular Innovations.

Techniques: Recombinant, Binding Assay, Concentration Assay, Software

Journal: Biochemistry

Article Title: High-affinity binding of LDL receptor–related protein 1 to matrix metalloprotease 1 requires protease:inhibitor complex formation

doi: 10.1021/acs.biochem.0c00442

Figure Lengend Snippet: Equilibrium binding constants for the interaction of proMMP-1 and TIMP-1 with LRP1 clusters.

Article Snippet: Recombinant human LRP1 cluster II, III, and IV Fc chimera proteins were obtained from Molecular Innovations.

Techniques: Binding Assay

Journal: Biochemistry

Article Title: High-affinity binding of LDL receptor–related protein 1 to matrix metalloprotease 1 requires protease:inhibitor complex formation

doi: 10.1021/acs.biochem.0c00442

Figure Lengend Snippet: Kinetic and equilibrium constants for the binding of MMP-1/TIMP-1 complexes to LRP1 ligand binding clusters.

Article Snippet: Recombinant human LRP1 cluster II, III, and IV Fc chimera proteins were obtained from Molecular Innovations.

Techniques: Binding Assay, Ligand Binding Assay

Journal: Biochemistry

Article Title: High-affinity binding of LDL receptor–related protein 1 to matrix metalloprotease 1 requires protease:inhibitor complex formation

doi: 10.1021/acs.biochem.0c00442

Figure Lengend Snippet: (A) Domain structure of MMP-9 with schematic representation of intact 92 kDa proMMP-9 and predicted fragment sizes by activation with MMP-3cd and APMA indicated. Purified TIMP-free MMP-9 (75 μg) was treated with either the MMP-3cd at 100 μM (B) or 2 mM APMA (C) for the indicated times. At each time point, 15 μg was removed for analysis by Coomassie protein staining or receptor blotting as indicated. Receptor binding was detected using monoclonal anti-LRP1 IgG 8G1 and goat anti-mouse IgG conjugated to HRP and visualized using chemiluminescence. MMP-9 is a 92 kDa zymogen. In the presence of either MMP-3cd or APMA, N-terminal cleavage of MMP-9 occurs yielding 86 and 82 kDa enzyme species (bracket). In the presence of APMA, the 82 kDa species undergoes C-terminal processing yielding a 68 kDa enzyme species (*).

Article Snippet: Recombinant human LRP1 cluster II, III, and IV Fc chimera proteins were obtained from Molecular Innovations.

Techniques: Activation Assay, Purification, Staining, Binding Assay

Journal: Biochemistry

Article Title: High-affinity binding of LDL receptor–related protein 1 to matrix metalloprotease 1 requires protease:inhibitor complex formation

doi: 10.1021/acs.biochem.0c00442

Figure Lengend Snippet: Full length LRP1 was immobilized on a CM5 sensor chip using an amine-reactive coupling process. (A) proMMP-9 was injected over the chip in increasing concentrations of ligand (11.1, 33.3, 100, and 300 nM). (B) proMMP-9/TIMP-1 complexes were injected over the chip in increasing concentrations of ligand (3.7, 11.1, 33.3, 100, and 300 nM). Fits of the experimental data (black lines) to a bivalent binding model are shown as blue lines. The data shown is a representative experiment from two independent experiments that were performed.

Article Snippet: Recombinant human LRP1 cluster II, III, and IV Fc chimera proteins were obtained from Molecular Innovations.

Techniques: Injection, Binding Assay

Journal: Nature Communications

Article Title: Brain-targeted drug delivery by manipulating protein corona functions

doi: 10.1038/s41467-019-11593-z

Figure Lengend Snippet: Characterization of ApoE functions in protein corona. a sLip or SP-sLip (100 μL, 10 mg mL −1 lipid) were incubated with BSA (10 μg mL −1 ) with or without rhApoE (10 μg mL −1 ) for 1 h at 37 °C, then recombinant human LRP1 (rhLRP1) was added and incubated for another 1 h at 37 °C. Protein coronas were isolated by centrifugation at 14k RCF, separated by SDS-PAGE and stained with silver. b SP-PEG3400-DSPE dissolved in methanol (0.1 μg per well) was coated in 96-well ELISA plate. After dry in room temperature, it was rinsed by cold PBS for three times. BSA (3%) was used to block for 2 h at room temperature. After thrice PBST rinses, anti-SP antibody (rabbit IgG, containing 0.1% BSA) was added and incubated for 1 h at 37 °C. Different concentrations of free SP (from 0 to 100 μg mL −1 ) or liposomes (sLip or SP-sLip, 0–5 mg mL −1 lipid) with or without plasma were incubated for another 1 h at 37 °C. HRP-anti-rabbit IgG was applied to detect the primary antibody using TMB kit. c sLip or SP-sLip were incubated with plasma for 1 h at 37 °C, then I 125 radiolabeled rhLRP1 was added in the mixture for another 1 h incubation. The radioactivity of liposomes after three cycles centrifugation and rinses was counted for detecting the absorbed rhLRP1. d Microscopic observation of cellular uptake of DiI-loaded sLip (sLip/DiI) or SP-sLip (SP-sLip/DiI) by bEnd.3 cells. sLip/DiI or SP-sLip/DiI was preincubated with PBS, rhApoE (10 μg mL −1 ) or mouse plasma for 1 h at 37 °C, then incubated with bEnd.3 cells for 4 h at 37 °C. Cellular uptake was detected by confocal laser scanning microscopy. Scale bar = 20 μm. e Quantitative analysis of DiI positive bEnd.3 cells by flow cytometry. Data are means ± SD ( n = 3), and analyzed with GraphPad Prism 6.0. *** p < 0.001 by student’s t -test

Article Snippet: Recombinant human LRP1 Cluster II Fc Chinera, Human IgG1 was from R&D system (Minneapolis, MN).

Techniques: Incubation, Recombinant, Isolation, Centrifugation, SDS Page, Staining, Enzyme-linked Immunosorbent Assay, Blocking Assay, Liposomes, Clinical Proteomics, Radioactivity, Confocal Laser Scanning Microscopy, Flow Cytometry

Journal: Cellular and Molecular Neurobiology

Article Title: Fc-Modified IVIG with Enhanced Blood–Brain Barrier Penetration Ameliorates Cognitive Deficits and Neuropathology in A53T α-Synuclein Transgenic Mice

doi: 10.1007/s10571-025-01626-1

Figure Lengend Snippet: Characterization of brain-targeting IVIG A and assessment of its blood–brain barrier permeability and bioactivity. A Schematic illustration of IVIG A construction. Angiopep-2 (Ang-2) peptides bearing NHNH₂ groups were chemically conjugated to the oxidized Asn297 glycan residues in the Fc domain of IVIG via sodium periodate-mediated coupling. B Dot blot analysis of IVIG A binding to LRP1. To evaluate the LRP1-binding activity of Ang-2 conjugated to IVIG, dot blot analysis was performed by spotting IVIG A and unmodified IVIG onto nitrocellulose membranes, followed by incubation with diluted recombinant LRP1 Cluster II protein. HRP-conjugated anti-human IgG antibody was used as a loading control. n = 6 (biological replicates, each value = mean of 3 independent experiments). C ELISA-based assessment of C1q binding to the Fc region. IVIG A or IVIG was incubated at concentrations ranging from 0.75 to 200 μg/mL on ELISA plates pre-coated with 2 μg/well of human C1q protein. Binding was detected using an HRP-conjugated anti-human IgG secondary antibody. n = 6 (biological replicates, each value = mean of 3 independent experiments). D Immunofluorescence analysis of IVIG A uptake by bEnd.3 cells. Cells were incubated with 500 μg/mL of IVIG A or unmodified IVIG for 2 h, then fixed and stained for human IgG (green), cell membranes (red), and nuclei (blue). Scale bar, 30 μm. E Quantification of intracellular hIgG signal intensity in bEnd.3 cells from D . n = 6 (biological replicates, each value = mean of 3 independent experiments). F – I Brain uptake and parenchymal distribution of IVIGᴬ vs IVIG in C57BL/6 mice. F Ex vivo fluorescence imaging of brains following systemic IVIG administration. C57BL/6 mice received a single tail vein injection of 25 mg Cy5-labeled IVIG A or IVIG. Brains were collected at 0, 1, 3, 6, 12, and 24 h post-injections following PBS transcardial perfusion. G Quantification of brain fluorescence intensity from F . n = 6 mice per group (biological replicates). H IVIG distribution in brain at the 3-h time point. Brains from the 3-h cohort in F were cryosectioned and immunostained for human IgG (green) and the vascular marker CD31 (red). Scale bar, 25 μm. I Quantification of hIgG fluorescence intensity in brain sections from H . n = 6 mice per group (biological replicates). J , K Anti-inflammatory activity of IVIGᴬ and IVIG in BV2 microglia. BV2 cells were pretreated in culture medium with IVIGᴬ or unmodified IVIG (500 μg/mL) for 2 h, followed by LPS stimulation. ELISA quantified tumor necrosis factor-α (TNF-α, J ) and interleukin-1β (IL-1β, K ) in the culture supernatants. n = 6 (biological replicates, each value = mean of 3 independent experiments). Data are presented as mean ± SD. Statistical tests were: Fig. 1 E , Welch’s ANOVA with Dunnett’s T3 post hoc; Fig. 1 I , unpaired two-tailed Student’s t-test; Fig. 1 J , Kruskal–Wallis with Bonferroni-adjusted Mann–Whitney U post hoc; Fig. 1 K , one-way ANOVA with Dunnett’s post hoc. Exact P values are annotated on the figures; when P < 0.01, values are reported as P < 0.01, otherwise exact values are shown.Full statistics (e.g., F, t, H/ U), degrees of freedom, and assumption tests (Shapiro–Wilk, Levene’s) are provided in the supplementary information

Article Snippet: One group was incubated with diluted LRP1 Cluster II protein (MedChemExpress, Cat# HY-P702520, 1 μg/mL), followed by goat anti-human LRP1 Cluster II antibody (Novus, Cat# AF2368-SP, 1:1,000), and then HRP-conjugated donkey anti-goat IgG antibody (Abcam, Cat# ab97120, RRID: AB_10679892, 1:5,000).

Techniques: Permeability, Glycoproteomics, Dot Blot, Binding Assay, Activity Assay, Incubation, Recombinant, Control, Enzyme-linked Immunosorbent Assay, Protein Binding, Immunofluorescence, Staining, Ex Vivo, Fluorescence, Imaging, Injection, Labeling, Marker, Two Tailed Test, MANN-WHITNEY

Journal: Cell

Article Title: Lrp1 is a host entry factor for Rift Valley fever virus.

doi: 10.1016/j.cell.2021.09.001

Figure Lengend Snippet: Figure 1. A pooled genome-scale CRISPR screen identifies Lrp1 and Lrp1-associated proteins RAP and Grp94 as critical proteins for RVFV infection (A) Schematic of the CRISPR/Cas9 screen in BV2 cells. (B) Light microscope images (43) of BV2 cells before infection and at 3 different time points post-infection. (C) At 18 dpi, surviving cells were reseeded into new flasks for reinfection on 19 dpi and imaged at 43 by light microscopy. (D) Volcano plot analysis of the BV2 screen results of surviving cells from the initial infection at an MOI 0.1. (E) Summary of key interactions that modulate Lrp1 surface presentation, including RAP and Grp94. See also Figure S1.

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies Rabbit anti-Lrp1 antibody Cell Signaling Cat# 64099, RRID: AB_2799654 Rabbit anti-His antibody Cell Signaling Cat# 2365 Mouse anti-tubulin antibody Sigma Aldrich Cat# T8328-200UL RRID: AB_1844090 Anti-RVFV clone 4-39-CC BEI Resources NR-43195 Anti-human IgG Fc (HRP) Abcam Cat# ab98624 RRID: AB_10673832 Anti-HA antibody SantaCruz sc-805 RRID: AB_631618 Anti-rabbit secondary antibody Alex Fluor-764 Jackson Immuno Research NC025445 Rabbit anti-RVFV N Genescript N/A Donkey anti-rabbit Cy3 secondary antibody Jackson Immuno Research Cat# 711-165-152 RRID: AB_2307443 Peroxidase-conjugated Donkey Anti-Mouse IgG Jackson Immuno Research Cat# 715-035-150 RRID: AB_2340770 Bacterial and virus strains E. coli BL21 (DE3) Novagen Cat# 69450 E. coli Stable3 Thermo Fisher Scientific Cat# C737303 RVFV ZH501 (reverse genetics generated) US CDC N/A RVFV MP12 GFP Zhang et al., 2018 N/A Adenovirus mCherry Vector Biolabs Cat# 1767 Adenodvirus mCherry Cre Vector Biolabs Cat# 1773 IAV PR8 strain Williams et al., 2018 N/A RSV GFP5 Viratree Cat# R125 VSV-RVFV-eGFP S. Whelan WashU N/A Lentivirus Addgene vectors Cat# 8454 RRID: Addgene_8454 Cat# 12260 RRID: Addgene_12260 VSV-eGFP S. Whelan WashU N/A Chemicals, peptides, and recombinant proteins RVFV Gn (aa 1-316) this manuscript N/A RVFV Gn (aa 1-410) Genscript N/A mRAPD3 this manuscript N/A mutant mRAPD3 this manuscript N/A human LRP1 CLII- Fc R & D Systems Cat# 2368-L2-050 human LRP1 CLIII- Fc R & D Systems Cat# 4824-L3-50 human LRP1 CLIV- Fc R & D Systems Cat# 5395-L4-050 human IgG1 Fc R & D Systems Cat# 110-HG-100 Control VP30 protein Xu et al., 2017 N/A Puromycin Sigma Aldrich Cat# P8833 Blasticidin GIBCO Cat# R21001 Lipofectamine 2000 Invitrogen Cat# 11668019 Surfen (GAG Inhibitor) Sigma Aldrich Cat# 362330 100MG Fetal Bovine Serum GIBCO Cat# F2442 Fetal Bovine Serum GIBCO Cat# F2442 HEPES Sigma Millipore Cat# 25-060-CI Sodium pyruvate Corning Cat# 25-000-CI Protein deglycosylation Mix II New England Biolabs Cat# P6044S DMEM Media GIBCO Cat# 11965084 (Continued on next page) Cell 184, 5163–5178.e1–e8, September 30, 2021 e1

Techniques: CRISPR, Infection, Light Microscopy

Journal: Cell

Article Title: Lrp1 is a host entry factor for Rift Valley fever virus.

doi: 10.1016/j.cell.2021.09.001

Figure Lengend Snippet: Figure 2. Lrp1 is essential for RVFV infection of BV2 cells (A) Western blot of BV2 Lrp1 knockout clones (Lrp1KO C3, Lrp1KO R1, Lrp1KO R2, Lrp1KO R4, Lrp1KO R5, and Lrp1KO R6), and partial knockout (Lrp1PKO R3) generated using either single gRNA or dual gRNA CRISPR/Cas9 approaches, as described in STAR Methods. (B) BV2 wild-type (WT) and Lrp1KO clones were infected with RVFV ZH501 at a MOI of 0.1. After 18 h, the cells were harvested for RNA extraction and subjected to RT-qPCR analysis. Data shown are viral RNA (vRNA) titers normalized to wild-type BV2 cells. (C) Microscopic images showing the WT and LRP1KO R4 cells infected with RVFV MP12GFP (MOI 5 for 6 h) in fluorescence images (top panels) and DAPI-stained images (bottom panels). Images were taken at 203 magnification (for quantification, refer to Figure S2H). (D) Flow cytometry of WT, Lrp1PKO R3, and Lrp1KO R4 cells infected with RVFV MP12GFP. (E) Corresponding analysis of flow cytometry histograms in (D). (F) Western blot of mouse embryonic fibroblasts (MEFs) from Lrp+/+ and Lrp1F/F mice infected with AdCre. (G) Representative flow cytometry of MEFs Lrp1+/+ and LrpF/F cells infected for 5 days with AdCre and then infected with RVFV-MP12GFP at MOI of 1 for 15 h. (H) Corresponding analysis of flow cytometry histogram data in (G).

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies Rabbit anti-Lrp1 antibody Cell Signaling Cat# 64099, RRID: AB_2799654 Rabbit anti-His antibody Cell Signaling Cat# 2365 Mouse anti-tubulin antibody Sigma Aldrich Cat# T8328-200UL RRID: AB_1844090 Anti-RVFV clone 4-39-CC BEI Resources NR-43195 Anti-human IgG Fc (HRP) Abcam Cat# ab98624 RRID: AB_10673832 Anti-HA antibody SantaCruz sc-805 RRID: AB_631618 Anti-rabbit secondary antibody Alex Fluor-764 Jackson Immuno Research NC025445 Rabbit anti-RVFV N Genescript N/A Donkey anti-rabbit Cy3 secondary antibody Jackson Immuno Research Cat# 711-165-152 RRID: AB_2307443 Peroxidase-conjugated Donkey Anti-Mouse IgG Jackson Immuno Research Cat# 715-035-150 RRID: AB_2340770 Bacterial and virus strains E. coli BL21 (DE3) Novagen Cat# 69450 E. coli Stable3 Thermo Fisher Scientific Cat# C737303 RVFV ZH501 (reverse genetics generated) US CDC N/A RVFV MP12 GFP Zhang et al., 2018 N/A Adenovirus mCherry Vector Biolabs Cat# 1767 Adenodvirus mCherry Cre Vector Biolabs Cat# 1773 IAV PR8 strain Williams et al., 2018 N/A RSV GFP5 Viratree Cat# R125 VSV-RVFV-eGFP S. Whelan WashU N/A Lentivirus Addgene vectors Cat# 8454 RRID: Addgene_8454 Cat# 12260 RRID: Addgene_12260 VSV-eGFP S. Whelan WashU N/A Chemicals, peptides, and recombinant proteins RVFV Gn (aa 1-316) this manuscript N/A RVFV Gn (aa 1-410) Genscript N/A mRAPD3 this manuscript N/A mutant mRAPD3 this manuscript N/A human LRP1 CLII- Fc R & D Systems Cat# 2368-L2-050 human LRP1 CLIII- Fc R & D Systems Cat# 4824-L3-50 human LRP1 CLIV- Fc R & D Systems Cat# 5395-L4-050 human IgG1 Fc R & D Systems Cat# 110-HG-100 Control VP30 protein Xu et al., 2017 N/A Puromycin Sigma Aldrich Cat# P8833 Blasticidin GIBCO Cat# R21001 Lipofectamine 2000 Invitrogen Cat# 11668019 Surfen (GAG Inhibitor) Sigma Aldrich Cat# 362330 100MG Fetal Bovine Serum GIBCO Cat# F2442 Fetal Bovine Serum GIBCO Cat# F2442 HEPES Sigma Millipore Cat# 25-060-CI Sodium pyruvate Corning Cat# 25-000-CI Protein deglycosylation Mix II New England Biolabs Cat# P6044S DMEM Media GIBCO Cat# 11965084 (Continued on next page) Cell 184, 5163–5178.e1–e8, September 30, 2021 e1

Techniques: Infection, Western Blot, Knock-Out, Clone Assay, Generated, CRISPR, RNA Extraction, Quantitative RT-PCR, Staining, Flow Cytometry, Cytometry

Journal: Cell

Article Title: Lrp1 is a host entry factor for Rift Valley fever virus.

doi: 10.1016/j.cell.2021.09.001

Figure Lengend Snippet: Figure 4. LRP1 binds RVFV glycoprotein Gn (A) LRP1 is comprised of four clusters, CLI, CLII, CLIII, and CLIV, and the cytoplasmic and transmembrane domains (left). Mini-domains of CLI, CLII, CLIII, and CLIV were generated as -Fc fusions (top right). Lentiviruses carrying either pLVX-empty vector or pLVX-expressing HA-tagged minidomains mini-LRP1 CLI, CLII, CLIII, and CLIV were also generated (bottom right).

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies Rabbit anti-Lrp1 antibody Cell Signaling Cat# 64099, RRID: AB_2799654 Rabbit anti-His antibody Cell Signaling Cat# 2365 Mouse anti-tubulin antibody Sigma Aldrich Cat# T8328-200UL RRID: AB_1844090 Anti-RVFV clone 4-39-CC BEI Resources NR-43195 Anti-human IgG Fc (HRP) Abcam Cat# ab98624 RRID: AB_10673832 Anti-HA antibody SantaCruz sc-805 RRID: AB_631618 Anti-rabbit secondary antibody Alex Fluor-764 Jackson Immuno Research NC025445 Rabbit anti-RVFV N Genescript N/A Donkey anti-rabbit Cy3 secondary antibody Jackson Immuno Research Cat# 711-165-152 RRID: AB_2307443 Peroxidase-conjugated Donkey Anti-Mouse IgG Jackson Immuno Research Cat# 715-035-150 RRID: AB_2340770 Bacterial and virus strains E. coli BL21 (DE3) Novagen Cat# 69450 E. coli Stable3 Thermo Fisher Scientific Cat# C737303 RVFV ZH501 (reverse genetics generated) US CDC N/A RVFV MP12 GFP Zhang et al., 2018 N/A Adenovirus mCherry Vector Biolabs Cat# 1767 Adenodvirus mCherry Cre Vector Biolabs Cat# 1773 IAV PR8 strain Williams et al., 2018 N/A RSV GFP5 Viratree Cat# R125 VSV-RVFV-eGFP S. Whelan WashU N/A Lentivirus Addgene vectors Cat# 8454 RRID: Addgene_8454 Cat# 12260 RRID: Addgene_12260 VSV-eGFP S. Whelan WashU N/A Chemicals, peptides, and recombinant proteins RVFV Gn (aa 1-316) this manuscript N/A RVFV Gn (aa 1-410) Genscript N/A mRAPD3 this manuscript N/A mutant mRAPD3 this manuscript N/A human LRP1 CLII- Fc R & D Systems Cat# 2368-L2-050 human LRP1 CLIII- Fc R & D Systems Cat# 4824-L3-50 human LRP1 CLIV- Fc R & D Systems Cat# 5395-L4-050 human IgG1 Fc R & D Systems Cat# 110-HG-100 Control VP30 protein Xu et al., 2017 N/A Puromycin Sigma Aldrich Cat# P8833 Blasticidin GIBCO Cat# R21001 Lipofectamine 2000 Invitrogen Cat# 11668019 Surfen (GAG Inhibitor) Sigma Aldrich Cat# 362330 100MG Fetal Bovine Serum GIBCO Cat# F2442 Fetal Bovine Serum GIBCO Cat# F2442 HEPES Sigma Millipore Cat# 25-060-CI Sodium pyruvate Corning Cat# 25-000-CI Protein deglycosylation Mix II New England Biolabs Cat# P6044S DMEM Media GIBCO Cat# 11965084 (Continued on next page) Cell 184, 5163–5178.e1–e8, September 30, 2021 e1

Techniques: Generated, Plasmid Preparation, Expressing

Journal: Journal of Virology

Article Title: LRP1 facilitates Jamestown Canyon virus infection of neurons

doi: 10.1128/jvi.01841-25

Figure Lengend Snippet: Lack of Lrp1 reduces JCV binding, internalization, and infection of murine cells. ( A, B ) Virus infectivity was determined by infecting WT and Lrp1 KO cells at an MOI of 0.1 with either ( A ) JCV or ( B ) ZIKV and quantifying viral RNA in the supernatant at 24 hpi (JCV) or 48 hpi (ZIKV). ( C, D ) JCV-N antigen (pink) counterstained with DAPI (blue). Slides were imaged at 10× using a Leica DMI8 inverted microscope. Scale bar = 250 µm. ( E, F ) Binding and internalization assays after surfen treatment using ( E ) JCV and ( F ) ZIKV as a control. Statistics were determined by two-way ANOVA with Dunnett’s multiple comparisons test ( A, E, F ) or Welch’s t -test ( B ). *, P < 0.05; ***, P < 0.001; ****, P < 0.0001; ns, no significance.

Article Snippet: The day of infection, human LRP1 Cluster II-Fc (R&D Systems, 2368-L2), LRP1 Cluster IV-Fc (R&D Systems, 5395-L4B), and human IgG Fc control (R&D Systems, 110-HG) were diluted to 20 μg/mL in DMEM containing 1% Pen/Strep and 1% L-Glut and serially diluted 1:2.

Techniques: Binding Assay, Infection, Virus, Inverted Microscopy, Control

Journal: Journal of Virology

Article Title: LRP1 facilitates Jamestown Canyon virus infection of neurons

doi: 10.1128/jvi.01841-25

Figure Lengend Snippet: CL IV of human LRP1 binds and neutralizes JCV. ( A–C ) FRNT assays were performed by mixing virus with increasing concentrations of LRP1 CL II -Fc, LRP1 CL IV -Fc, or an Fc control protein prior to infection of Vero E6 cells. Twenty-four hours later, foci were visualized by immunostaining, imaged using a Cytation 5, and quantified using ImageJ. Statistics were determined by two-way ANOVA with Dunnett’s multiple comparisons test. ( D ) Biolayer interferometry sensograms displaying association and dissociation of JCV virions to immobilized LRP1 CL IV -Fc-His (blue), CL II -Fc-His (red), or recombinant human IgG1 Fc (gray). Data were baseline subtracted using sensors in buffer alone. BLI experiments were performed in triplicate. *, P < 0.05; **, P < 0.01; ****, P < 0.0001; ns, no significance.

Article Snippet: The day of infection, human LRP1 Cluster II-Fc (R&D Systems, 2368-L2), LRP1 Cluster IV-Fc (R&D Systems, 5395-L4B), and human IgG Fc control (R&D Systems, 110-HG) were diluted to 20 μg/mL in DMEM containing 1% Pen/Strep and 1% L-Glut and serially diluted 1:2.

Techniques: Virus, Control, Infection, Immunostaining, Recombinant

Journal: Journal of Virology

Article Title: LRP1 facilitates Jamestown Canyon virus infection of neurons

doi: 10.1128/jvi.01841-25

Figure Lengend Snippet: Replication kinetics of JCV in primary rat neurons. Primary rat neurons were infected with JCV at the indicated MOI. ( A ) Viral RNA or ( B ) infectious virus was quantified at 24, 36, 48, and 60 hpi time points. ( C ) Infected or mock-infected coverslips were stained for JCV-N (pink) and βIII-tubulin (green) and counterstained with DAPI (blue). Slides were imaged at 20× using a Nikon A1 confocal microscope. Scale bar = 250 µm. ( D ) Immunofluorescent microscopy of neurons after 4 days of culture. Coverslips were stained for Lrp1 (green) and counterstained with DAPI (blue). Slides were imaged at 10× using a Leica DMI8 inverted microscope. Scale bar = 250 µm.

Article Snippet: The day of infection, human LRP1 Cluster II-Fc (R&D Systems, 2368-L2), LRP1 Cluster IV-Fc (R&D Systems, 5395-L4B), and human IgG Fc control (R&D Systems, 110-HG) were diluted to 20 μg/mL in DMEM containing 1% Pen/Strep and 1% L-Glut and serially diluted 1:2.

Techniques: Infection, Virus, Staining, Microscopy, Inverted Microscopy

Journal: Journal of Virology

Article Title: LRP1 facilitates Jamestown Canyon virus infection of neurons

doi: 10.1128/jvi.01841-25

Figure Lengend Snippet: Pre-treatment with a high-affinity Lrp1 binding protein reduces JCV infection. Primary rat neurons were pre-treated with different concentrations of mRAP D3 for 45 min, followed by infection with JCV ( A ) or ZIKV MR766 ( B ) at an MOI of 0.1. Supernatant was collected for quantification of virus at 24 hpi (JCV) or 48 hpi (ZIKV). ( C ) Coverslips were stained for JCV-N (pink) and βIII-tubulin (green) and counterstained with DAPI (blue). Slides were imaged at 10× using a Leica DMI8 inverted microscope. Scale bar = 250 µm. Statistics were determined by two-way ANOVA with Dunnett’s multiple comparisons test. *, P < 0.05; ****, P < 0.0001; ns, no significance.

Article Snippet: The day of infection, human LRP1 Cluster II-Fc (R&D Systems, 2368-L2), LRP1 Cluster IV-Fc (R&D Systems, 5395-L4B), and human IgG Fc control (R&D Systems, 110-HG) were diluted to 20 μg/mL in DMEM containing 1% Pen/Strep and 1% L-Glut and serially diluted 1:2.

Techniques: Binding Assay, Infection, Virus, Staining, Inverted Microscopy

Journal: Journal of Virology

Article Title: LRP1 facilitates Jamestown Canyon virus infection of neurons

doi: 10.1128/jvi.01841-25

Figure Lengend Snippet: Specificity of Lrp1 for JCV entry. ( A ) BV2 WT and LRP1 KO cells infected with either JCV, OROV, or RVFV. ( B ) BV2 WT cells pre-treated with 0 or 10 µg/mL of mRAP D3 , followed by infection. ( C ) BV2 Lrp1 KO cells pre-treated with 0 or 10 µg/mL of mRAP D3 , followed by infection. Statistics were determined by two-way ANOVA with Dunnett’s multiple comparisons test. *, P < 0.05; ***, P < 0.001; ****, P < 0.0001; ns, no significance.

Article Snippet: The day of infection, human LRP1 Cluster II-Fc (R&D Systems, 2368-L2), LRP1 Cluster IV-Fc (R&D Systems, 5395-L4B), and human IgG Fc control (R&D Systems, 110-HG) were diluted to 20 μg/mL in DMEM containing 1% Pen/Strep and 1% L-Glut and serially diluted 1:2.

Techniques: Infection

Journal: Sensors (Basel, Switzerland)

Article Title: Applications of Surface Plasmon Resonance (SPR) for the Characterization of Nanoparticles Developed for Biomedical Purposes

doi: 10.3390/s121216420

Figure Lengend Snippet: SPR study of protein corona. ( a – c ): PMMA NPs were preincubated in human plasma for 1 hour, precipitated by centrifugation, resuspended in buffer and injected for 3 min onto chip surfaces immobilizing LRP-1 (a), anti-ApoE Ab (b) or anti-HSA Ab (c). Purple lines show the corresponding sensorgrams. As internal controls we injected NPs samples preincubated in buffer (blue sensorgrams) or sample obtained, with the same procedure (centrifugation and resuspension) from plasma alone (red sensorgrams). Black sensorgrams were obtained as difference between the purple and the red sensorgrams, to highlight the signal specifically due to proteins adsorbed to NPs during the incubation in plasma. ( d – f ): Black sensorgrams were obtained as described above. Resuspended NPs samples underwent further centrifugation and supernatants were injected (gray sensorgrams). See text for details.

Article Snippet: For NP corona studies, recombinant human low-density lipoprotein receptor-related protein-1 (LRP-1) cluster IV Fc chimera (R&D Systems, Minneapolis, MN, USA), anti-apolipoprotein E antibody (ApoE-Ab, Abcam, Cambridge, UK) and anti-human albumin antibody (HSA-Ab, GeneTex, Irvine, CA, USA) were immobilized in parallel-flow channels of a GLC sensor chip (BioRad) using amine-coupling chemistry.

Techniques: Centrifugation, Injection, Incubation