Journal: medRxiv

Article Title: Polymorphism in IFNAR contributes to glucocorticoid response and outcome in ARDS and COVID-19

doi: 10.1101/2022.03.10.22272123

Figure Lengend Snippet: (A ) STAT1 expression in the lung after 4-day culture in the presence of IFN beta with or without hydrocortisone (HC). ( B) pSTAT1 expression in the same specimens as in A. ( C) Example photomicrographs showing higher STAT2 expression in a TT patient than in a CT patient and the effect of HC on its nuclear translocation. Most STAT2 remains in the cytoplasm of the CT patients, whereas nuclear expression is prominent in the TT patient. Indicated insets are shown in the bottom row. Arrows. ( D ) Combined results of all patients noting that two CT samples are excluded in the data as the patients were already under glucocorticoid treatment at the time of sample acquisition. Ns, not significant; *P<0.05; **P<0.01; and ***P<0.001

Article Snippet: The first stage antibodies were anti-alpha chain of the IFN alpha/beta receptor (St John’s Laboratory STJ112765) that was used 1:2000 and 1:5000 and anti-Stat1 (1:400, 9175S), anti-pStat1(1:100, 9167S) and anti-Stat2 (1:200, 72604S) all from Cell Signalling.

Techniques: Expressing, Translocation Assay

Journal: Molecular cancer research : MCR

Article Title: RASEF is a novel diagnostic biomarker and a therapeutic target for lung cancer.

doi: 10.1158/1541-7786.MCR-12-0685-T

Figure Lengend Snippet: Figure 1. RASEF expression in tumor tissues and cell lines. A, expression of RASEF in 12 clinical lung cancers (T; 4 clinical lung ADC, 4 clinical lung SCC, and 4 clinical SCLC) and corresponding normal lung tissues (N) detected by semiquantitative RT-PCR analysis. B, expression of RASEF in 22 lung cancer cell lines and a bronchial epithelial cell line BEAS-2B detected by semiquantitative RT-PCR analysis. ASC indicates lung adenosquamous cell carcinoma; LCC, large cell carcinoma. C, Western blot analysis of RASEF protein using anti-RASEF antibody. IB, immunoblotting. D, expression and subcellular localization of endogenous RASEF protein in RASEF-positive and RASEF-negative lung cancer cell lines, and bronchial epithelial cells. RASEF was stained mainly at the cytoplasm in A549 and NCI-H2170 cells, whereas no staining was observed in DMS114 and bronchial epithelia–derived BEAS-2B cell lines.

Article Snippet: The cells were then incubated overnight at 4 C with a rabbit polyclonal antibody to RASEF (Catalog No. 11569-1-AP, Proteintech Group, Inc.) diluted in PBS containing 1% bovine serum albumin (BSA).

Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Western Blot, Staining, Derivative Assay

Journal: Molecular cancer research : MCR

Article Title: RASEF is a novel diagnostic biomarker and a therapeutic target for lung cancer.

doi: 10.1158/1541-7786.MCR-12-0685-T

Figure Lengend Snippet: Figure 4. Enhanced phosphorylation of ERK1/2 by RASEF in lung cancer cells. A, expression of MAPK signal molecules and their phosphorylation levels in DMS114 cells transfected with RASEF expression vector or mock plasmid. B, expression of MAPK signal molecules and their phosphorylation levels in NCI-H2170 cells transfected with siRNAs for RASEF (si-RASEF#2) or control siRNAs (si-LUC). C and D, expression levels of downstream target genes of MAPK cascade were regulated by RASEF expression in lung cancer cells. Total RNA from BEAS-2B and DMS114 cells transfected with RASEF expression vector or mock plasmid (C) and A549 and NCI-H2170 cells transfected with siRNAs for RASEF (si-RASEF#2) or control siRNAs (si-LUC; D) were subjected to reverse-transcription reaction, followed by PCR reaction to evaluate the expression levels of CCND1, CCNB1, and CDKN1A transcription. Western blotting with antiphosphorylated ERK1/2 antibody was conducted to confirm the change of ERK1/2 phosphorylation according to RASEF expression.

Article Snippet: The cells were then incubated overnight at 4 C with a rabbit polyclonal antibody to RASEF (Catalog No. 11569-1-AP, Proteintech Group, Inc.) diluted in PBS containing 1% bovine serum albumin (BSA).

Techniques: Phospho-proteomics, Expressing, Transfection, Plasmid Preparation, Control, Reverse Transcription, Western Blot

Journal: Molecular cancer research : MCR

Article Title: RASEF is a novel diagnostic biomarker and a therapeutic target for lung cancer.

doi: 10.1158/1541-7786.MCR-12-0685-T

Figure Lengend Snippet: Figure 5. Identification of ERK1/2-interacting sites on RASEF. A, interaction of endogenous RASEF with endogenous ERK1/2. The immunoprecipitates obtained using anti-RASEF antibody were subjected to Western blotting with anti-ERK1/2 antibody. B, schematic representation of various partial constructs of RASEF expression vector. C and D, determination of the ERK1/2-interacting regions on RASEF by immunoprecipitation experiments using DMS114 cells transfected with vectors expressing partial RASEF protein. COOH-terminal part of RASEF (codons 520–575) was likely to be ERK1/2-interacting region.

Article Snippet: The cells were then incubated overnight at 4 C with a rabbit polyclonal antibody to RASEF (Catalog No. 11569-1-AP, Proteintech Group, Inc.) diluted in PBS containing 1% bovine serum albumin (BSA).

Techniques: Western Blot, Construct, Expressing, Plasmid Preparation, Immunoprecipitation, Transfection

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: Frontiers in Molecular Biosciences

Article Title: Accumulated ROS Activates HIF-1α-Induced Glycolysis and Exerts a Protective Effect on Sensory Hair Cells Against Noise-Induced Damage

doi: 10.3389/fmolb.2021.806650

Figure Lengend Snippet: Gene-specific primers in this study.

Article Snippet: The following antibodies were used: anti-SOD1 (rabbit monoclonal, ab51254, Abcam), anti-SOD2 (rabbit monoclonal, ab68155, Abcam), anti-Catalase (rabbit polyclonal, 21260-1-AP, Proteintech), anti-GPx1 (rabbit monoclonal, ab108429, Abcam), anti-GLUT1 (mouse monoclonal, ab238050, Abcam), anti-HK2 (rabbit monoclonal, ab209847, Abcam), anti-ENO1 (rabbit monoclonal, ab227978, Abcam), anti-PDK1 (rabbit monoclonal, ab202468, Abcam), anti-LDHA (mouse monoclonal, 66287-1-Ig, Proteintech), anti-HIF-1α (rabbit monoclonal, ab179483, Abcam; mouse monoclonal, 66730-1-Ig, Proteintech), anti-β-Tubulin (rabbit polyclonal, 10068-1-AP, Proteintech), anti-Myosin VIIa (rabbit monoclonal, ab155984, Abcam), and 4-HNE (mouse monoclonal, ab48506, Abcam).

Techniques:

Journal: Endocrine Connections

Article Title: The emerging role of the molecular marker p27 in the differential diagnosis of adrenocortical tumors

doi: 10.1530/EC-13-0025

Figure Lengend Snippet: Percentage area stained for the different immunohistochemical markers (mean± s.e.m .) in the different groups.

Article Snippet: Endogenous peroxidase was blocked with 3% hydrogen peroxide in methanol, followed by incubation with normal serum for 30 min. Then, the samples were incubated overnight at 4 °C with the appropriate primary antibodies: rabbit anti-human polyclonal antibodies to StAR (HPA023644; 1:100; Atlas Antibodies, Stockholm, Sweden), IGF2 (ab9574; 1:100; Abcam, Cambridge, UK), MDM2 (ab15471; 1:100; Abcam), and IGF1R (ab39675; 1:100; Abcam), The samples were then incubated with secondary antibodies at 1:200 dilution (polyclonal swine anti-rabbit, Dako, Glostrup, Denmark) for 30 min, followed by incubation with an avidin–biotin peroxidase complex (1:100; Vector Laboratories, Inc., Peterborough, UK) for 30 min. Diaminobenzidine was used as the chromogen and hematoxylin as the nuclear counterstain.

Techniques: Staining, Immunohistochemical staining

Journal: American journal of transplantation : official journal of the American Society of Transplantation and the American Society of Transplant Surgeons

Article Title: Nox2 is a mediator of chronic CsA nephrotoxicity

doi: 10.1111/j.1600-6143.2012.04081.x

Figure Lengend Snippet: NRK52E cells were cultured in the presence of CsA (1μM and 10μM) or TGF-β1 (20 ng/ml) for 48 hours. Immunoblot analyses demonstrated a significant increase in phosphorylated (activated) p38MAPK, Smad3, and NFκB signaling molecules, as well as Nox2 and α-SMA protein levels in response to CsA and TGF-β1. The blot shown is a representative sample of three replicate studies, summarized in the bar graph.

Article Snippet: We used the following antibodies: Nox2/gp91 phox (1:100-250), BD Biosciences, San Diego, CA, 611414, Phospho p38 (1:500), BD 612288, Phospho NFκB (1:250), Abcam, Cambridge, MA, ab28849, α-SMA (1:2000), Sigma, St. Louis, MO, A2547, β-Actin (1:7500), US Biological, Marblehead, MA, A0760-40, Fibronectin (1:400), Abcam, ab23750, HNE (mouse, 15ug/mL), Abcam, ab48506, GAPDH (1:5000), Abcam, ab8245, Phospho Smad3 (1:500) Cell Signaling, Danvers, MA, 9520, HNE (rabbit, 1:1000), Abcam, ab4654.

Techniques: Cell Culture, Western Blot