Journal: The Journal of Cell Biology

Article Title: Membrane nanoclusters of FcγRI segregate from inhibitory SIRPα upon activation of human macrophages

doi: 10.1083/jcb.201608094

Figure Lengend Snippet: SIRPα and FcγRI are arranged in discrete nanoclusters at macrophage surfaces. (A and B) TIRF and dSTORM images of SIRPα (A) and FcγRI (B) at the surface of human macrophages seeded onto PLL- (nonactivated, top) or hIgG-coated slides (bottom) for 10 min and stained with fluorescently labeled specific antibodies. Bars, 5 µm. Regions delineated by white squares are zoomed-in and shown with corresponding density maps (pseudocolor scale), thresholded binary maps and Ripley’s K analysis of the molecules in the selected regions. Bars, 1 µm. L(r)-r represents the degree of clustering relative to simulated random distributions, indicated by the 99% confidence intervals (CIs); r is the radial scale. (C–E) Nanocluster areas (C), density (D), and percentage of localizations in nanoclusters (E) for SIRPα and FcγRI under nonactivating (black) or hIgG-activating (gray) conditions were calculated by subjecting dSTORM data to spatial point-pattern analysis and thresholding. Each symbol represents the median of several 5 × 5 µm regions from the same cell. Horizontal lines and error bars represent mean ± SD. Data are from a minimum of 30 cells from three independent donors. ns, not significant; ****, P < 0.0001; two-tailed t test assuming unequal variance. (F and G) Label-density variation analysis for SIRPα (F) and FcγRI (G) yields characteristic normalized ρ/η curves for clustered proteins. Cells were stained with anti–SIRPα-AF647 (F) or anti–FcγRI-AF488 (G) at different labeling concentrations and imaged by dSTORM. Each data point represents a single cell, color-coded by antibody concentration used for labeling. Red lines indicate reference curves for a random distribution of molecules.

Article Snippet: Primary monoclonal antibodies used for microscopy were anti-SIRPα (clone 4C7; AbD Serotec) conjugated in-house with AF647 (Invitrogen), anti–FcγRI-AF488 (clone 10.1; BioLegend), anti-FcγRII (clone FLI8.26; BD) conjugated in-house with Atto488 (Invitrogen) or AF647, and anti–PTPN6(Tyr536)-AF647 (Bioss).

Techniques: Staining, Labeling, Two Tailed Test, Concentration Assay

Journal: The Journal of Cell Biology

Article Title: Membrane nanoclusters of FcγRI segregate from inhibitory SIRPα upon activation of human macrophages

doi: 10.1083/jcb.201608094

Figure Lengend Snippet: SIRPα and FcγRI nanoclusters are constitutively associated in nonactivated human macrophages but segregate upon activation with hIgG. (A) TIRF and dSTORM images showing FcγRI (green) and SIRPα (red) at the surface of human macrophages incubated for 10 min on slides coated with PLL (nonactivated, top) or hIgG (middle) and stained with anti–FcγRI-AF488 and anti–SIRPα-AF647 mAbs. Bars, 5 µm. Regions outlined by the white squares (middle column) are shown enlarged (right columns) with relative fluorescence intensity profiles along the white lines. Bars, 1 µm. As a positive control, macrophages seeded onto PLL-coated slides were stained with anti–FcγRI-AF488 mAb followed by anti–mouse IgG1-AF647 secondary antibody (bottom). (B) CBC histograms of the single-molecule distributions of the colocalization parameter for SIRPα and FcγRI in cells seeded onto PLL- (gray) or hIgG-coated (red) slides for 10 min or for positive control data (green). Data are from a minimum of 30 cells from three independent donors. Bars represent mean ± SD. (C) Nearest-neighbor (NN) analysis from data shown in (B). Each symbol represents the median NN of all paired single-molecule localizations from one cell. Horizontal lines and error bars represent mean ± SD. ****, P < 0.0001; two-tailed t test assuming unequal variance. (D) Histogram distributions of the NND between the centroids of nanoclusters from one channel and the centroid of their nearest neighbor from the second channel (≥ 20,000 clusters from a minimum of 10 cells per condition) from cells seeded onto PLL- (light gray), hIgG-coated (light red) slides, or positive control data (green). Corresponding simulated data are also shown, in which the centroid positions of SIRPα nanoclusters in both nonactivating (dark gray) and hIgG-activating conditions (dark red) were randomized within the cell area.

Article Snippet: Primary monoclonal antibodies used for microscopy were anti-SIRPα (clone 4C7; AbD Serotec) conjugated in-house with AF647 (Invitrogen), anti–FcγRI-AF488 (clone 10.1; BioLegend), anti-FcγRII (clone FLI8.26; BD) conjugated in-house with Atto488 (Invitrogen) or AF647, and anti–PTPN6(Tyr536)-AF647 (Bioss).

Techniques: Activation Assay, Incubation, Staining, Fluorescence, Positive Control, Two Tailed Test

Journal: The Journal of Cell Biology

Article Title: Membrane nanoclusters of FcγRI segregate from inhibitory SIRPα upon activation of human macrophages

doi: 10.1083/jcb.201608094

Figure Lengend Snippet: SIRPα and the low-affinity Fc receptor, FcγRII, are segregated on a nanometer scale. (A) TIRF and dSTORM images showing FcγRII (green) and SIRPα (red) at the surface of human macrophages incubated for 10 or 30 min on slides coated with PLL (nonactivated) or hIgG and stained with anti–FcγRII-AF488 and anti–SIRPα-AF647 mAbs. Bars, 5 µm. In each condition, regions outlined by the white squares (middle column) are shown enlarged (right column) with relative fluorescence intensity profiles along the white lines. Bars, 1 µm. (B) CBC histograms of the single-molecule distributions of the colocalization parameter for SIRPα and FcγRII in cells seeded onto PLL- or hIgG-coated slides for 10 (light gray and dark gray, respectively) or 30 min (light red and dark red, respectively) or for positive control data (green). The positive control data in this figure is the same as in . Data are from a minimum of 30 cells from three independent donors. Bars represent mean ± SD. (C) NND analysis from data shown in B. Each symbol represents the median NND of all paired single-molecule localizations from one cell. Horizontal lines and error bars represent mean ± SD. ns, not significant; **, P < 0.01; ***, P < 0.001; one-way analysis of variance (ANOVA) with Tukey’s post-hoc test. (D) Histogram distributions of the NND between the centroids of nanoclusters from one channel and the centroid of their nearest neighbor from the second channel (≥20,000 clusters from a minimum of 10 cells per condition). a.u. arbitrary units; NN, nearest neighbor; PC, positive control.

Article Snippet: Primary monoclonal antibodies used for microscopy were anti-SIRPα (clone 4C7; AbD Serotec) conjugated in-house with AF647 (Invitrogen), anti–FcγRI-AF488 (clone 10.1; BioLegend), anti-FcγRII (clone FLI8.26; BD) conjugated in-house with Atto488 (Invitrogen) or AF647, and anti–PTPN6(Tyr536)-AF647 (Bioss).

Techniques: Incubation, Staining, Fluorescence, Positive Control

Journal: The Journal of Cell Biology

Article Title: Membrane nanoclusters of FcγRI segregate from inhibitory SIRPα upon activation of human macrophages

doi: 10.1083/jcb.201608094

Figure Lengend Snippet: FcγRs reorganize into concentric rings upon activation. (A) TIRF images of FcγRI (top) and FcγRII (bottom) at the surface of human macrophages incubated for 10 or 30 min on slides coated with PLL (nonactivated) or hIgG and stained with fluorescently labeled specific antibodies. Bars, 10 µm. (B) TIRF and dSTORM images of FcγRI (green) and FcγRII (red) at the surface of macrophages incubated for 10 or 30 min on slides coated with PLL or hIgG and stained with anti–FcγRI-AF488 and anti–FcγRII-AF647 mAbs. Bars, 5 µm. Regions outlined by the white squares (middle column) are shown enlarged (right column) with relative fluorescence intensity profiles along the white lines. Bars, 1 µm. (C) CBC histograms of the single-molecule distributions of the colocalization parameter for FcγRI and FcγRII in cells seeded onto PLL- or hIgG-coated slides for 10 (light gray and dark gray, respectively) or 30 min (light red and dark red, respectively) or for positive control data (green). The positive control data in this figure are the same as in . Data are from a minimum of 10 cells from three independent donors. Bars represent mean ± SD. (D) NND analysis from data shown in C. Each symbol represents the median NND of all paired single-molecule localizations from one cell. Horizontal lines and error bars represent mean ± SD. ns, not significant; **, P < 0.01; ****, P < 0.0001; one-way ANOVA with Tukey’s post-hoc test. (E) Histogram distributions of the NND between the centroids of nanoclusters from one channel and the centroid of their nearest neighbor from the second channel (≥20,000 clusters from a minimum of 10 cells per condition). a.u., arbitrary units; NN, nearest neighbor; PC, positive control.

Article Snippet: Primary monoclonal antibodies used for microscopy were anti-SIRPα (clone 4C7; AbD Serotec) conjugated in-house with AF647 (Invitrogen), anti–FcγRI-AF488 (clone 10.1; BioLegend), anti-FcγRII (clone FLI8.26; BD) conjugated in-house with Atto488 (Invitrogen) or AF647, and anti–PTPN6(Tyr536)-AF647 (Bioss).

Techniques: Activation Assay, Incubation, Staining, Labeling, Fluorescence, Positive Control

Journal: The Journal of Cell Biology

Article Title: Membrane nanoclusters of FcγRI segregate from inhibitory SIRPα upon activation of human macrophages

doi: 10.1083/jcb.201608094

Figure Lengend Snippet: Specific activation of FcγRI is required for its reorganization into concentric rings and segregation from SIRPα nanoclusters. (A and B) TIRF (bars, 10 µm) and dSTORM (bars, 5 µm) images showing FcγRI (green) and SIRPα (red) at the surface of human macrophages incubated for 10 (A) or 30 min (B) on slides coated with hIgG1 or hIgG2 and stained with anti–FcγRI-AF488 and anti–SIRPα-AF647 mAbs. In each condition, regions outlined by the white squares (middle column) are shown enlarged (right column) with relative fluorescence intensity profiles along the white lines. Bars, 1 µm. (C) CBC histograms of the single-molecule distributions of the colocalization parameter for FcγRI and SIRPα in cells seeded onto hIgG1- or hIgG2-coated slides for 10 (light gray and dark gray, respectively) or 30 min (light red and dark red, respectively). Data are from a minimum of 30 cells from three independent donors. Bars represent mean ± SD. (D) NND analysis from data shown in C. Each symbol represents the median NND of all paired single-molecule localizations from one cell. Horizontal lines and error bars represent mean ± SD. ns, not significant; **, P < 0.01; ****, P < 0.0001; one-way ANOVA with Tukey’s post-hoc test. (E) Histogram distributions of the NND between the centroids of nanoclusters from one channel and the centroid of their nearest neighbor from the second channel (≥20,000 clusters from a minimum of 10 cells per condition). a.u., arbitrary units; NN, nearest neighbor; PC, positive control.

Article Snippet: Primary monoclonal antibodies used for microscopy were anti-SIRPα (clone 4C7; AbD Serotec) conjugated in-house with AF647 (Invitrogen), anti–FcγRI-AF488 (clone 10.1; BioLegend), anti-FcγRII (clone FLI8.26; BD) conjugated in-house with Atto488 (Invitrogen) or AF647, and anti–PTPN6(Tyr536)-AF647 (Bioss).

Techniques: Activation Assay, Incubation, Staining, Fluorescence, Positive Control

Journal: The Journal of Cell Biology

Article Title: Membrane nanoclusters of FcγRI segregate from inhibitory SIRPα upon activation of human macrophages

doi: 10.1083/jcb.201608094

Figure Lengend Snippet: Rearrangement of macrophage surface receptors triggered by mobile hIgG. (A) TIRF images of FcγRI at the surface of human macrophages incubated for 10 min on SLBs loaded with streptavidin (nonactivating) or with streptavidin-hIgG (activating) and stained with a fluorescently labeled specific antibody. Two example images are shown for each condition. Bars, 10 µm. (B) dSTORM images of FcγRI (green) and SIRPα (red) at the surface of macrophages seeded as in A and stained with anti–FcγRI-AF488 and anti–SIRPα-AF647 mAbs. Bars, 5 µm. Regions outlined by the white squares are shown enlarged with relative fluorescence intensity profiles along the white lines. Bars, 1 µm. (C–E) Nanocluster areas (C), density (D), and percentage of localizations in nanoclusters (E) for SIRPα and FcγRI under nonactivating (black) or hIgG-activating (gray) conditions. Each symbol represents the median of several 5 × 5 µm regions from the same cell. Horizontal lines and error bars represent mean ± SD. Data are from a minimum of 30 cells from two independent experiments. ns, not significant; *, P < 0.05; ****, P < 0.0001; two-tailed t test assuming unequal variance. (F) CBC histograms of the single-molecule distributions of the colocalization parameter for SIRPα and FcγRI in cells seeded as in A. Data are from a minimum of 30 cells from two independent experiments. Bars represent mean ± SD. (G) NND analysis from data shown in F. Each symbol represents median NND of all paired single-molecule localizations from one cell. Horizontal lines and error bars represent mean ± SD. ****, P < 0.0001; two-tailed t test assuming unequal variance. (H) Histogram distributions of the NND between the centroids of nanoclusters from one channel and the centroid of their nearest neighbor from the second channel (≥10,000 clusters from a minimum of 10 cells per condition) from cells seeded onto control nonactivating (light gray) or hIgG-loaded activating (light red) SLBs.

Article Snippet: Primary monoclonal antibodies used for microscopy were anti-SIRPα (clone 4C7; AbD Serotec) conjugated in-house with AF647 (Invitrogen), anti–FcγRI-AF488 (clone 10.1; BioLegend), anti-FcγRII (clone FLI8.26; BD) conjugated in-house with Atto488 (Invitrogen) or AF647, and anti–PTPN6(Tyr536)-AF647 (Bioss).

Techniques: Incubation, Staining, Labeling, Fluorescence, Two Tailed Test

Journal: The Journal of Cell Biology

Article Title: Membrane nanoclusters of FcγRI segregate from inhibitory SIRPα upon activation of human macrophages

doi: 10.1083/jcb.201608094

Figure Lengend Snippet: Ligation of SIRPα impairs the reorganization of surface FcγRI. (A) Human macrophages were incubated for 24 h in wells coated with PLL, 20 µg/ml of hCD47, or with increasing concentrations of hCD47 in the presence of 10 µg/ml of hIgG, as indicated. M-CSF release was assessed by ELISA. Bars represent mean ± SD from three donors. Each color represents one individual donor. (B) TIRF images of FcγRI at the surface of human macrophages incubated for 10 min on slides coated with hCD47 or hCD47 plus hIgG and stained with fluorescently labeled specific antibody. Bars, 10 µm. (C) TIRF and dSTORM images showing FcγRI (green) and SIRPα (red) at the surface of human macrophages incubated for 10 min on slides coated with hCD47 (top) or hCD47 plus hIgG (bottom) and stained with anti–FcγRI-AF488 and anti–SIRPα-AF647 mAbs. Bars, 5 µm. In each condition, regions outlined by the white squares (middle column) are shown enlarged (right column) with relative fluorescence intensity profiles along the white lines. Bars, 1 µm. (D and G) CBC histograms of the single-molecule distributions of the colocalization parameter for FcγRI and SIRPα (D) and for FcγRI and pSHP-1 Y536 (G) in cells seeded onto slides coated with PLL (light gray), hCD47 (light red), hCD47 plus hIgG (dark red), or hIgG (dark gray) for 10 (D) or 5 min (G). Data are from a minimum of 30 cells from three independent donors. Bars represent mean ± SD. (E and H) NND analysis from data shown in D and G, respectively. Each symbol represents the median NND of all paired single-molecule localizations from one cell. Horizontal lines and error bars represent mean ± SD. ns, not significant; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001; one-way ANOVA with Tukey’s post-hoc test. (F and I) Histogram distributions of the NND between the centroids of nanoclusters from one channel and the centroid of their nearest neighbor from the second channel (≥20,000 clusters from a minimum of 10 cells per condition). Graphs compare colocalization between FcγRI and SIRPα (F) and FCγRI and pSHP-1 Y536 (I). a.u., arbitrary units; NN, nearest neighbor; PC, positive control.

Article Snippet: Primary monoclonal antibodies used for microscopy were anti-SIRPα (clone 4C7; AbD Serotec) conjugated in-house with AF647 (Invitrogen), anti–FcγRI-AF488 (clone 10.1; BioLegend), anti-FcγRII (clone FLI8.26; BD) conjugated in-house with Atto488 (Invitrogen) or AF647, and anti–PTPN6(Tyr536)-AF647 (Bioss).

Techniques: Ligation, Incubation, Enzyme-linked Immunosorbent Assay, Staining, Labeling, Fluorescence, Positive Control

Journal: The Journal of Cell Biology

Article Title: Membrane nanoclusters of FcγRI segregate from inhibitory SIRPα upon activation of human macrophages

doi: 10.1083/jcb.201608094

Figure Lengend Snippet: Segregation and reorganization of FcγRI is dependent on the actin cytoskeleton and formins, but not myosin II. (A) TIRF image of FcγRI (white; bars, 20 µm) and dSTORM images (bars, 5 µm) of FcγRI (green) and SIRPα (red) at the surface of human macrophages pretreated with 1 µM latrunculin A, 0.5 µM jasplakinolide, 10 µM blebbistatin or 10 µM SMIFH2. Cells were then seeded onto slides coated with PLL (nonactivated) or hIgG for 10 min, and stained with anti-FcγRI-AF488 and anti-SIRPα-AF647 mAbs. In each condition, regions outlined by the white squares (middle column) are shown enlarged (right column). Bars, 1 µm. (B) CBC histograms of the single-molecule distributions of the colocalization parameter for FcγRI and SIRPα in cells pretreated with drugs as indicated and seeded onto slides coated with PLL (gray) or hIgG (latrunculin A [Lat A], dark gray; jasplakinolide [Jasp], red; SMIFH2, green; or blebbistatin [Bleb], blue) for 10 min. Data are from a minimum of 30 cells per condition from three independent donors. Bars represent mean ± SD. (C) NND analysis from data shown in B. Each symbol represents the median NND of all paired single-molecule localizations from one cell. Horizontal lines and error bars represent mean ± SD. ns, not significant; **, P < 0.01; ****, P < 0.0001; two-tailed t test assuming unequal variance. (D) Histogram distributions of the NND between the centroids of nanoclusters from one channel and the centroid of their nearest neighbor from the second channel (≥20,000 clusters from a minimum of 10 cells per condition). (E–G) Nanocluster areas (E), density (F), and percentage of localizations in nanoclusters (G) for SIRPα and FcγRI under nonactivating (black) or hIgG-activating (gray) conditions after pretreatment of cells with blebbistatin or DMSO control. Each symbol represents the median of several 5 × 5 µm regions from the same cell. Horizontal lines and error bars represent mean ± SD. Data are from a minimum of 30 cells from three independent donors. ns, not significant; *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001; two-tailed t test assuming unequal variance. NN, nearest neighbor.

Article Snippet: Primary monoclonal antibodies used for microscopy were anti-SIRPα (clone 4C7; AbD Serotec) conjugated in-house with AF647 (Invitrogen), anti–FcγRI-AF488 (clone 10.1; BioLegend), anti-FcγRII (clone FLI8.26; BD) conjugated in-house with Atto488 (Invitrogen) or AF647, and anti–PTPN6(Tyr536)-AF647 (Bioss).

Techniques: Staining, Two Tailed Test

Journal: The Journal of Cell Biology

Article Title: Membrane nanoclusters of FcγRI segregate from inhibitory SIRPα upon activation of human macrophages

doi: 10.1083/jcb.201608094

Figure Lengend Snippet: Src-family kinase signaling, but not Syk or PI3K signaling, is indispensable for reorganization of macrophage surfaces. (A) Immunoblots of phosphorylated AKT in nonactivated (PLL) or hIgG-activated human macrophages pretreated with vehicle (DMSO), as a control, 10 µM PP2 (left), 100 µM piceatannol (PCT; middle), or 1 µM wortmannin (Wort; right). Blots represent two independent experiments. (B) TIRF image of FcγRI (white; bars, 20 µm) and dSTORM images (bars, 5 µm) of FcγRI (green) and SIRPα (red) at the surface of human macrophages incubated with vehicle (DMSO), PP2, PCT, or Wort, pretreated as in A. Cells were then seeded onto slides coated with PLL (nonactivated) or hIgG for 10 min and stained with anti–FcγRI-AF488 and anti–SIRPα-AF647 mAbs. In each condition, regions outlined by the white squares (middle column) are shown enlarged (right column). Bars, 1 µm. (C) CBC histograms for FcγRI and SIRPα in cells pretreated as in A and seeded onto slides coated with PLL (gray) or hIgG (DMSO, dark gray; PP2, red; PCT, green; and Wort, blue) for 10 min, as indicated. Data are from a minimum of 30 cells from three independent donors. Bars show mean ± SD. (D) NND analysis from data shown in C. Each symbol represents the median NND of all paired single-molecule localizations from one cell. Horizontal lines and error bars represent mean ± SD. ns, not significant; ****, P < 0.0001; one-way ANOVA with Tukey’s post-hoc test. (E) Histogram distributions of the NND between the centroids of nanoclusters from one channel and the centroid of their nearest neighbor from the second channel (≥20,000 clusters from a minimum of 10 cells per condition).

Article Snippet: Primary monoclonal antibodies used for microscopy were anti-SIRPα (clone 4C7; AbD Serotec) conjugated in-house with AF647 (Invitrogen), anti–FcγRI-AF488 (clone 10.1; BioLegend), anti-FcγRII (clone FLI8.26; BD) conjugated in-house with Atto488 (Invitrogen) or AF647, and anti–PTPN6(Tyr536)-AF647 (Bioss).

Techniques: Western Blot, Incubation, Staining

Journal: Journal of materials chemistry. B

Article Title: Attenuation of Thrombin-Mediated Fibrin Formation via Changes in Fibrinogen Conformation Induced by Reaction with S- nitroso- N -acetylpenicillamine, but not S- nitrosoglutathione

doi: 10.1039/C8TB02103A

Figure Lengend Snippet: Western immunoblot analysis of fibrinogen subunits, Aa, Bb and γ after 1 h exposure with SNAP, NAP and SNP in absence and presence of thrombin. (A), Representative immunoblots. Aliquots (15 μl) of samples were separated on 12% polyacrylamide gels, protein transferred to poly(vinyl-difluoride) (PVDF) membranes and then immunostained with mouse anti-human alpha fibrinogen, rabbit anti-human beta fibrinogen and goat anti-human gamma fibrinogen antibodies (all diluted 1:200). After membrane washings, the membranes were incubated with the secondary antibodies chicken anti-mouse-AF488, chicken anti-rabbit-AF647 and donkey anti-goat-AF555 (all diluted 1:500) for 1 h. After two 10 min washes the blots were imaged in a fluorescence imager (Bio-Rad VersaDoc MP4000 Hercules, CA). Molecular weight markers are shown on left with positions of the relevant fibrinogen chains and Aαγ dimers indicated on the right. (B) Western analysis of samples in (A), after the 1 h incubation, were quantified by densitometry using Quanity One imaging software (Bio-Rad Hercules, CA). The specific Aαγ dimer formation was calculated as the ratio (density) of the Aαγ dimer band over the sum of dimers plus monomers. The data was collected after 1 h in presence of thrombin. The ratio data are means + SEM (n=3). * = p<0.05, SNAP or NAP vs SNP.

Article Snippet: After membrane washings, the membranes were incubated with the secondary antibodies chicken anti-mouse-AF488, chicken anti-rabbit-AF647 and donkey anti-goat-AF555 (all diluted 1:500) for 1 h. After two 10 min washes the blots were imaged in a fluorescence imager (Bio-Rad VersaDoc MP4000 Hercules, CA).

Techniques: Western Blot, Incubation, Fluorescence, Molecular Weight, Imaging, Software

Journal: RSC Advances

Article Title: Close-packed silane nanodot arrays by capillary nanostamping coupled with heterocyclic silane ring opening †

doi: 10.1039/c9ra03440d

Figure Lengend Snippet: Capillary nanostamping of N -aminoethyl-aza-2,2-dimethyl-4-methylsilacyclopentane in toluene onto glass slides. (a) Reaction scheme of the ring opening reaction with the surface hydroxyl groups. (b) and (c) TIRFM images of aminosilane nanodot arrays stamped onto glass slides after binding the dye ATTO 647 NHS to the terminal amine groups of the silanes. (b) 4 th and (c) 10 th consecutive stamping cycle under ambient conditions without reinking. The raw TIRFM images were deconvolved using the Huygens software. The scale bars correspond to 5 μm. Insets are zoom-in regions of 5 × 5 μm 2 .

Article Snippet: The fluorescent dyes ATTO 647 NHS and ATTO 655 maleimide were obtained from ATTO-TEC GmbH, Germany.

Techniques: Binding Assay, Software

Journal: Nature

Article Title: Designed endocytosis-inducing proteins degrade targets and amplify signals.

doi: 10.1038/s41586-024-07948-2

Figure Lengend Snippet: Fig. 1 | Design strategies for endocytosis-triggering EndoTags. a, Schema of designed endocytosis mechanisms. Top, design of binding to constitutively cycling receptors at sites that do not overlap with binding sites for natural ligands to avoid competition. Middle, design of binders that trigger endocytosis by eliciting conformational changes in the receptor. The EndoTag binds at two distinct epitopes on the target and actively triggers the conformational change. Bottom, designed endocytosis via receptor clustering. The multivalent EndoTag clusters multiple copies of the target receptor and induces endocytosis. b, Design strategy for sortilin48 and TfR49 EndoTags. c, Cellular uptake of 100 nM AF647-labelled Sort_EndoTags, TfR-EndoTags or LHDB22 scaffold control for 2 h in U-251MG cells. Data were normalized to the 100 nM AF647-labelled LHDB group (no endocytosis). MFI, mean fluorescence intensity. d, Confocal imaging of Sort_EndoTag (red) and lysosomal marker (green, AF488-labelled LysoTracker) after 24 h incubation in U-251MG cells. e, Design strategy for

Article Snippet: Anti-LAMP2A antibody (Abcam ab18528), goat anti-rabbit- IgG Alexa FluorTM 488 secondary antibody (Thermo Fisher A-11034), anti PD-L1 (sc-518027), anti beta-actin (sc-47778), goat anti-mouse IgG H&L (HRP) (Abcam, ab205719); 800CW goat-anti-mouse or goatanti-rabbit (LI-COR 926-32211), rabbit anti-CTLA4 E1V6T Cell Signaling Technologies (96399) Validation Alexa Fluor® 647 anti-human EGFR Antibody (Biolegend, 352918): Verified Reactivity to Human, FC - Quality tested PE anti-human CD222 (IGF2R) Recombinant Antibody (Biolegend, 364204): Verified Reactivity to Human, ICFC, FC - Quality tested EGFR Monoclonal Antibody (clone 199.12, Invitrogen, AHR5072): Target Species: Human, applications for Immunocytochemistry, Immunofluorescence, Immunoprecipitation, Western Blot IGF2R Antibody Alexa647 (clone 2G11, Novus, NB300-514AF647): Reactivity Hu, Mu, Rt, Bv, Pm; Applications WB, ELISA, Flow, ICC/IF, IHC, IP, CyTOF-ready Human PD-L1 Alexa Fluor® 647-conjugated Antibody (Rndsystems, FAB1562R): Species Reactivity Human, Detects human PD-L1/B7H1 in direct ELISAs.

Techniques: Binding Assay, Control, Fluorescence, Imaging, Marker, Incubation

Journal: Nature

Article Title: Designed endocytosis-inducing proteins degrade targets and amplify signals.

doi: 10.1038/s41586-024-07948-2

Figure Lengend Snippet: Fig. 3 | Clearance of soluble proteins by IGF2R pLYTACs. a, Schema for the use of soluble pLYTACs with IGF_EndoTags. b, Cellular uptake of LHDB–AF647 via LHDA–IGF_EndoTags in Jurkat cells. Cells were incubated with 33 nM LHDB– AF647 with or without 1 μM LHDA–IGF_EndoTags for 24 h, washed twice with cold PBS and analysed by flow cytometry. c, Remaining supernatant LHDB– AF647 levels in Jurkat cells. Jurkat cells were incubated with 100 nM LHDB– AF647 with or without 500 nM LHDA–IGF_EndoTags. At timepoints 24 h and 48 h, the cells were pelleted down, and IgG in the supernatant was quantified using a Neo2 plate reader. IgG level was normalized to the IgG-alone control group. d, Cellular uptake of IgG–AF647 via protein G–IGF_EndoTags in K562 cells. Cells were incubated with 33 nM IgG–AF647 with or without 1 μM protein G–IGF_EndoTag3 for 24 h, washed twice with cold PBS and analysed by flow cytometry. The fold change in MFI was calculated by normalizing to the

Article Snippet: Anti-LAMP2A antibody (Abcam ab18528), goat anti-rabbit- IgG Alexa FluorTM 488 secondary antibody (Thermo Fisher A-11034), anti PD-L1 (sc-518027), anti beta-actin (sc-47778), goat anti-mouse IgG H&L (HRP) (Abcam, ab205719); 800CW goat-anti-mouse or goatanti-rabbit (LI-COR 926-32211), rabbit anti-CTLA4 E1V6T Cell Signaling Technologies (96399) Validation Alexa Fluor® 647 anti-human EGFR Antibody (Biolegend, 352918): Verified Reactivity to Human, FC - Quality tested PE anti-human CD222 (IGF2R) Recombinant Antibody (Biolegend, 364204): Verified Reactivity to Human, ICFC, FC - Quality tested EGFR Monoclonal Antibody (clone 199.12, Invitrogen, AHR5072): Target Species: Human, applications for Immunocytochemistry, Immunofluorescence, Immunoprecipitation, Western Blot IGF2R Antibody Alexa647 (clone 2G11, Novus, NB300-514AF647): Reactivity Hu, Mu, Rt, Bv, Pm; Applications WB, ELISA, Flow, ICC/IF, IHC, IP, CyTOF-ready Human PD-L1 Alexa Fluor® 647-conjugated Antibody (Rndsystems, FAB1562R): Species Reactivity Human, Detects human PD-L1/B7H1 in direct ELISAs.

Techniques: Incubation, Flow Cytometry, Control