Journal: Investigative Ophthalmology & Visual Science

Article Title: Normalization of Wound Healing and Diabetic Markers in Organ Cultured Human Diabetic Corneas by Adenoviral Delivery of c-Met Gene

doi: 10.1167/iovs.09-4569

Figure Lengend Snippet: Antibodies

Article Snippet: Positive In Source/Reference c-Met total Rabbit pAb sc-161 IHC, WB Santa Cruz Biotechnology c-Met total Rabbit pAb sc-10 IHC, WB Santa Cruz Biotechnology c-Met total Rabbit pAb sc-8307 WB * Santa Cruz Biotechnology c-Met total Rabbit pAb 71-8000 Zymed c-Met extracellular Mouse mAb MAB358 WB R&D Systems c-Met extracellular Mouse mAb 05-238 IHC Millipore p-c-Met (Tyr1003) Rabbit pAb ab61024 IHC Abcam p-c-Met (Tyr1234/Tyr1235) Rabbit pAb 44887G WB * Invitrogen p-c-Met (Tyr1230/Tyr1234/Tyr1235) Rabbit pAb 44888G Invitrogen HGF Rabbit pAb sc-7949 IHC Santa Cruz Biotechnology Nidogen-1 Mouse mAb A9 IHC Kabosova et al. 37 Nidogen-1 Mouse mAb MAB2570 IHC R&D Systems Nidogen-2 Rabbit pAb 1080 IHC Kabosova et al. 37 Laminin γ1 chain Rat mAb A5 IHC Kabosova et al. 37 Integrin α 3 β 1 Mouse mAb MAB1992 IHC Millipore Akt Mouse mAb 610860 WB BD Transduction Labs p-Akt (Ser472/Ser473) Mouse mAb 550747 BD Pharmingen p-Akt (Ser473) Rabbit pAb sc-7985-R IHC, WB Santa Cruz Biotechnology p-Akt (Ser473) Rabbit pAb 9271 IHC, WB Cell Signaling p-Akt (Thr308) Rabbit mAb 2965 Cell Signaling ERK1/2 Rabbit mAb 4695 IHC, WB Cell Signaling p-ERK1/2 (Thr202/Tyr204) Rabbit pAb sc-101760 Santa Cruz Biotechnology p-ERK1/2 (Tyr 204) Mouse mAb sc-7383 Santa Cruz Biotechnology p-ERK1/2 (Thr202/Tyr204) Rabbit mAb 4370 WB Cell Signaling p-ERK1/2 (Thr185/Thr202) Rabbit pAb ab4819 IHC Abcam p-ERK1/2 (Thr183/Tyr185) Mouse mAb ab50011 Abcam p38 MAPK Rabbit pAb 9212 WB Cell Signaling p-p38 (Thr180/Tyr182) Rabbit pAb AB3828 WB * Millipore p-p38 (Thr180/Tyr182) Rabbit mAb 9215 WB Cell Signaling p-p38 (Thr180/Tyr182) Mouse mAb 612280 BD Transduction Labs p-p38 (Thr180/Tyr182) Mouse mAb ab50012 IHC Abcam p85α PI3 kinase Mouse mAb sc-56939 Santa Cruz Biotechnology p-p85α PI3 kinase (Tyr508) Goat pAb sc-12929 Santa Cruz Biotechnology p-EGFR (Tyr845) Rabbit pAb 44-784G IHC Invitrogen p-EGFR (Tyr845) Rabbit mAb 2342-1 Epitomics ZO-1 Rabbit pAb 40-2300 IHC † Invitrogen Claudin-1 Rabbit pAb 51-9000 IHC † Invitrogen Ki-67 Mouse mAb sc-101861 IHC ‡ Santa Cruz Biotechnology Activated caspase-3 Rabbit pAb G7481 IHC Promega β-Actin Mouse mAb A5316 WB Sigma-Aldrich Open in a separate window Santa Cruz Biotechnology, Santa Cruz, CA; Zymed, South San Francisco, CA; R&D Systems, Minneapolis, MN; Millipore, Billerica, MA; Abcam, Cambridge, MA; Invitrogen, Carlsbad, CA; BD Transduction Laboratories, Lexington, KY; BD Pharmingen, San Diego, CA; Cell Signaling, Beverly, MA; Epitomics, Burlingame, CA; Promega, Madison, WI; Sigma-Aldrich, St. Louis, MO. mAb, monoclonal antibody; pAb, polyclonal antibody; IHC, immunohistochemistry; WB, Western blot; p-, phosphorylated.

Techniques: Western Blot, Transduction, Immunohistochemistry

Journal: iScience

Article Title: Analysis of myocardial cellular gene expression during pressure overload reveals matrix based functional intercellular communication

doi: 10.1016/j.isci.2022.103965

Figure Lengend Snippet: Endothelial, fibroblast and cardiomyocyte cell purification from mouse hearts (A) Scheme on the experimental procedures to isolate the indicated cell types from mouse hearts. (B) Heatmap of cell marker gene expression in RNA from isolated cardiomyocytes (CM), endothelial cells (EC) or fibroblasts (FB) after sham or the indicated time point after TAC surgery. (C) Cardiac endothelial cells and fibroblasts were isolated from mouse hearts and stained for the endothelial markers CD31 and CD102, for the leukocyte marker CD45, and the fibroblast marker Mefsk4. Subsequently, flow cytometric analyses were performed and representative results are shown here. The numbers indicated in each quadrant indicates the percentage of cells localized in that particular quadrant. (D) RNA from the different cell types after sham or 1 and 8 weeks after TAC was subjected to RNA sequencing. The differences in overall gene expression patterns were visualized by a principal component analysis.

Article Snippet: CD45 Antibody, anti-mouse , Miltenyi Biotec , Clone 30F11.

Techniques: Purification, Marker, Gene Expression, Isolation, Staining, RNA Sequencing

Journal: iScience

Article Title: Analysis of myocardial cellular gene expression during pressure overload reveals matrix based functional intercellular communication

doi: 10.1016/j.isci.2022.103965

Figure Lengend Snippet:

Article Snippet: CD45 Antibody, anti-mouse , Miltenyi Biotec , Clone 30F11.

Techniques: Purification, Plasmid Preparation, Blocking Assay, Magnetic Beads, Recombinant, Clinical Proteomics, Protease Inhibitor, cDNA Synthesis, SYBR Green Assay, Enzyme-linked Immunosorbent Assay, Software

Journal: Frontiers in Microbiology

Article Title: Wheat germ cell-free system-based production of hemagglutinin-neuraminidase glycoprotein of human parainfluenza virus type 3 for generation and characterization of monoclonal antibody

doi: 10.3389/fmicb.2014.00208

Figure Lengend Snippet: Epitope mapping for generated antibodies. (A) Schematic diagram of seven deletion mutants of HPIV3-HN for epitope mapping. These proteins were produced as N-terminal biotinylated protein by wheat germ cell-free system. (B) Schematic diagram of the AlphaScreen assay used to detect the binding of MAb to full-length or deletion mutants of HPIV3-HN. The interaction between antibodies and recombinant proteins was monitored by AlphaScreen with protein A-conjugated acceptor beads and streptavidin-coated donor. Upon excitation at 680 nm, singlet oxygen molecules were produced from the donor beads, which reacted with the acceptor beads, resulting in light emission that was measured between 520 and 620 nm. (C) In AlphaScreen assay, the binding activity was measured as the level of the AlphaScreen luminescence signal. Error bars represent standard deviations from three independent experiments. (D) The biotinylated-full-length HN, its deletion mutants and GST proteins were separated by SDS-PAGE and transferred to PVDF membrane, followed by immunoblotting with Strepavidin-HRP andibody (left panel) and indicated MAb (right panel). (E) Summary of properties of selected MAbs. Immunoglobulin isotyping was carried out with mouse monoclonal antibody isotyping test kit.

Article Snippet: Isotype determination was performed with the mouse MAb isotyping test kit (Bio-Rad, Hercules, CA, USA) according to the manufacturer’s protocol.

Techniques: Generated, Produced, Amplified Luminescent Proximity Homogenous Assay, Binding Assay, Recombinant, Activity Assay, SDS Page, Western Blot

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: T-helper cell diversity in experimental autoimmune encephalomyelitis and Multiple Sclerosis

doi: 10.4049/jimmunol.1501097

Figure Lengend Snippet: IL-12 and IL-23 polarized CD4+ T cells are both capable of mediating ON. (A-C) C57BL/6 mice were immunized with MOG35-55 emulsified in CFA. Ten to fourteen days later, draining lymph node cells were harvested and cultured for 96 hours with MOG35-55 and either recombinant IL-12 or IL-23 to generate Th1 and Th17 cells, respectively. (A) Intracellular staining and flow cytometric analysis of cultured cells at 96 hours, gating on the CD3+CD4+ population. (B, C) Following culture, CD4+ T cells were transferred into naïve syngenic recipients. Mice in each group were euthanized at day 9 post-transfer, the day after clinical EAE onset. Mononuclear cells isolated from optic nerves (ON) and spinal cords (SC) were assessed for CD4+ T cell cytokine production (B) and the percent of CD4+ T cells and CD11b+ myeloid cells among CD45+ leukocytes (C) by flow cytometry. Data are representative of three independent experiments with at least 3 mice per group.

Article Snippet: The following antibodies were used for flow cytometry or immunohistochemistry: rat anti-MBP (Millipore); mouse anti-unphosphorylated neurofilament-H (SMI-32, Covance); CD45 (Serotec), Brn3a (Santa Cruz); Alexa Fluor 594 goat anti-mouse IgG and Alexa Fluor 488 goat anti-rat IgG (Life Technologies); FITC–anti-MHCII, FITC–anti-B220, PE–anti-CD45 (Ly5), PE–anti-CD8α, PE–anti-CD4, PE–anti–GM-CSF, PECy7–anti-CD11b, and PECy7–anti-CD4; PerCpCy5.5–anti-Ly6C, PerCPCy5.5–anti-CD3ε, and PerCPCy5.5–anti–IL-17A (e-Bioscience); BD Biosciences: allophycocyanin–anti-CD45.2, FITC–anti-CD44, allophycocyanin cy7–anti-Ly6G, allophycocyanin cy7–anti-CD45.1, and allophycocyanin cy7–anti–IFN-γ (BD Biosciences).

Techniques: Cell Culture, Recombinant, Staining, Isolation, Flow Cytometry

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: T-helper cell diversity in experimental autoimmune encephalomyelitis and Multiple Sclerosis

doi: 10.4049/jimmunol.1501097

Figure Lengend Snippet: Axonopathy is an early feature of both Th1- and Th17-mediated ON. (A–D) Th1 and Th17 adoptive transfer recipients were injected i.o. with fluorochrome-conjugated cholera toxin-B (red) at onset of clinical EAE. Optic nerves were harvested 24 hours later. Longitudinal sections were stained for the pan-leukocyte marker, CD45 (green) to demarcate areas of inflammation. (C) Confocal microscopy revealed a swelling at the tip of a transected axon (asterisk). (D) The frequency of axonal swellings was compared with the intensity of CD45 staining and there was no significant difference. (E–G) Cross- sections of optic nerves harvested from mice with Th17- (E) and Th1- (F) mediated ON were stained for unphosphorylated neurofilament-H (SMI-32, green; DAPI, blue; bars=50 μm). Magnified views of the insets are shown in adjacent panels (bars=25 μm). (G) SMI-32 staining of a healthy nerve (bar= 50 μm). (H–J) Representative cross-sections of optic nerves obtained from mice with Th17- (H) or Th1-(I) mediated ON, or from naïve mice (J), were stained with toludine blue. Broken lines delineate representative areas with normal-appearing axons, asteriscs indicate swollen axons and arrowheads point to empty myelin sheaths (bars= 25 μm).

Article Snippet: The following antibodies were used for flow cytometry or immunohistochemistry: rat anti-MBP (Millipore); mouse anti-unphosphorylated neurofilament-H (SMI-32, Covance); CD45 (Serotec), Brn3a (Santa Cruz); Alexa Fluor 594 goat anti-mouse IgG and Alexa Fluor 488 goat anti-rat IgG (Life Technologies); FITC–anti-MHCII, FITC–anti-B220, PE–anti-CD45 (Ly5), PE–anti-CD8α, PE–anti-CD4, PE–anti–GM-CSF, PECy7–anti-CD11b, and PECy7–anti-CD4; PerCpCy5.5–anti-Ly6C, PerCPCy5.5–anti-CD3ε, and PerCPCy5.5–anti–IL-17A (e-Bioscience); BD Biosciences: allophycocyanin–anti-CD45.2, FITC–anti-CD44, allophycocyanin cy7–anti-Ly6G, allophycocyanin cy7–anti-CD45.1, and allophycocyanin cy7–anti–IFN-γ (BD Biosciences).

Techniques: Adoptive Transfer Assay, Injection, Staining, Marker, Confocal Microscopy

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: T-helper cell diversity in experimental autoimmune encephalomyelitis and Multiple Sclerosis

doi: 10.4049/jimmunol.1501097

Figure Lengend Snippet: Axon loss and faulty nerve conduction occur in both Th1- and Th17-mediated ON. (A–D) Optic nerves harvested immediately before the peak of EAE were stained with antibodies against SMI32 (green) and CD45 (red). Representative cross-sections of nerves from mice with Th17- (A) and Th1- (B) mediated ON, as well as from a naïve mouse (B), are shown (bars = 100 μm and 25 μm, inset) and quantified (D). (E) Brn3a+ RGCs were counted in retinas harvested from naïve mice, and from ON mice at serial time points post-transfer (t-test). (F) Representative traces of CAPs of optic nerves acutely isolated from naïve and ON mice. CAP velocities (G, I) and amplitudes (H, J) were averaged over seven to twelve nerves per group. Data presented as mean ±S.E.M. (**p<0.01, ***p<0.001, ****p<0.0001; Mann-Whitney) Scale bars: A, B 50μm, insets 25μm; C 25μm.

Article Snippet: The following antibodies were used for flow cytometry or immunohistochemistry: rat anti-MBP (Millipore); mouse anti-unphosphorylated neurofilament-H (SMI-32, Covance); CD45 (Serotec), Brn3a (Santa Cruz); Alexa Fluor 594 goat anti-mouse IgG and Alexa Fluor 488 goat anti-rat IgG (Life Technologies); FITC–anti-MHCII, FITC–anti-B220, PE–anti-CD45 (Ly5), PE–anti-CD8α, PE–anti-CD4, PE–anti–GM-CSF, PECy7–anti-CD11b, and PECy7–anti-CD4; PerCpCy5.5–anti-Ly6C, PerCPCy5.5–anti-CD3ε, and PerCPCy5.5–anti–IL-17A (e-Bioscience); BD Biosciences: allophycocyanin–anti-CD45.2, FITC–anti-CD44, allophycocyanin cy7–anti-Ly6G, allophycocyanin cy7–anti-CD45.1, and allophycocyanin cy7–anti–IFN-γ (BD Biosciences).

Techniques: Staining, Isolation, MANN-WHITNEY

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: T-helper cell diversity in experimental autoimmune encephalomyelitis and Multiple Sclerosis

doi: 10.4049/jimmunol.1501097

Figure Lengend Snippet: Stable Th17 cells induce ON. (A) Intracellular cytokine production by CD4+ T cells derived from MOG-immunized IL-12KO mice, after 96 hours of culture with MOG35-55 and recombinant IL-23. (B) Intracellular cytokine production by CD4+ T cells derived from MOG-immunized IL-12KO donors after infiltrating the spinal cords and optic nerves of IL-12KO hosts. (C) Clinical courses of IL-12KO mice injected with myelin-specific CD4+ Th17 cells derived from IL-12KO donors versus WT mice injected with WT effector Th17 cells. The data shown is pooled from 3 experiments with n=10 WT and n=17 IL12KO host mice per group. (D) The percentage of CD4+ T cells and CD11b+ myeloid cells among CD45+ leukocytes infiltrating the optic nerve and spinal cord of IL-12KO hosts shortly after the onset of clinical EAE. (E) Contiguous sections of an optic nerve obtained from an IL-12KO host at clinical EAE onset and stained with CD45 (red, top left panel) or SMI-32 (green). (F) A representative section stained with toludine blue (bars= 50μm in D, 25μm in E). Areas with normal appearing axons are outlined, asteriscs indicate swollen axons and arrowheads point to empty myelin sheaths. (G–I) CAPs were measured in acutely isolated optic nerves from the IL-12KO recipients of stable Th17 cells at clinical EAE onset. (G) Representative wave-forms of optic nerve CAPs from a naïve IL-12KO mouse and an IL-12KO adoptive transfer recipient with acute ON. The data were averaged over seven to nine nerves per group. Data are presented as mean ±S.E.M. (*p<0.05, **p<0.01; Mann-Whitney)

Article Snippet: The following antibodies were used for flow cytometry or immunohistochemistry: rat anti-MBP (Millipore); mouse anti-unphosphorylated neurofilament-H (SMI-32, Covance); CD45 (Serotec), Brn3a (Santa Cruz); Alexa Fluor 594 goat anti-mouse IgG and Alexa Fluor 488 goat anti-rat IgG (Life Technologies); FITC–anti-MHCII, FITC–anti-B220, PE–anti-CD45 (Ly5), PE–anti-CD8α, PE–anti-CD4, PE–anti–GM-CSF, PECy7–anti-CD11b, and PECy7–anti-CD4; PerCpCy5.5–anti-Ly6C, PerCPCy5.5–anti-CD3ε, and PerCPCy5.5–anti–IL-17A (e-Bioscience); BD Biosciences: allophycocyanin–anti-CD45.2, FITC–anti-CD44, allophycocyanin cy7–anti-Ly6G, allophycocyanin cy7–anti-CD45.1, and allophycocyanin cy7–anti–IFN-γ (BD Biosciences).

Techniques: Derivative Assay, Recombinant, Injection, Staining, Isolation, Adoptive Transfer Assay, MANN-WHITNEY

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: T-helper cell diversity in experimental autoimmune encephalomyelitis and Multiple Sclerosis

doi: 10.4049/jimmunol.1501097

Figure Lengend Snippet: Bona Fide Th1 cells are capable of inducing ON. (A) Intracellular cytokine production by CD4+ T cells derived from MOG-immunized IL-23KO mice, after 96 hours of culture with MOG35-55 and recombinant IL-12. (B) Intracellular cytokine production by CD4+ T cells derived from MOG-immunized IL-23KO donors after infiltrating the spinal cords and optic nerves of IL-23KO hosts. (C) Clinical courses of IL-23KO mice injected with myelin-specific CD4+ Th1 cells derived from IL-23KO donors versus WT mice injected with WT effector Th17 cells. The data was pooled from 3 experiments with n=9 WT and n=22 IL-23KO hosts (*p<0.05, **p<0.01; Holm-Sidak multiple t-tests). (D) The percentage of CD4+ T cells and CD11b+ myeloid cells among CD45+ leukocytes infiltrating the optic nerve and spinal cord. (E) Contiguous sections of a representative optic nerve obtained from an IL-23KO host at clinical EAE onset and stained with CD45 (red, left panel) or SMI-32 (green, right panels). (F) A representative section stained with toluidine blue. (bars= 50μm in E, 25μm in insets and in F). Examples of areas with normal appearing axons are outlined, asterisks mark examples of swollen axons and arrowheads point to myelin sheaths left behind by degenerated axons. (G–I) CAPs were measured in optic nerves from the IL-23KO recipients of Th1 cells at clinical EAE onset. The data were averaged over seven to nine nerves per group. (G) Representative wave-forms. CAP velocities (H) and amplitudes (I) were measured in eight to twelve nerves per group. Data are presented as mean ± S.E.M. (*p<0.05, **p<0.01; Mann-Whitney)

Article Snippet: The following antibodies were used for flow cytometry or immunohistochemistry: rat anti-MBP (Millipore); mouse anti-unphosphorylated neurofilament-H (SMI-32, Covance); CD45 (Serotec), Brn3a (Santa Cruz); Alexa Fluor 594 goat anti-mouse IgG and Alexa Fluor 488 goat anti-rat IgG (Life Technologies); FITC–anti-MHCII, FITC–anti-B220, PE–anti-CD45 (Ly5), PE–anti-CD8α, PE–anti-CD4, PE–anti–GM-CSF, PECy7–anti-CD11b, and PECy7–anti-CD4; PerCpCy5.5–anti-Ly6C, PerCPCy5.5–anti-CD3ε, and PerCPCy5.5–anti–IL-17A (e-Bioscience); BD Biosciences: allophycocyanin–anti-CD45.2, FITC–anti-CD44, allophycocyanin cy7–anti-Ly6G, allophycocyanin cy7–anti-CD45.1, and allophycocyanin cy7–anti–IFN-γ (BD Biosciences).

Techniques: Derivative Assay, Recombinant, Injection, Staining, MANN-WHITNEY

Journal: eLife

Article Title: ADAM17-dependent signaling is required for oncogenic human papillomavirus entry platform assembly

doi: 10.7554/eLife.44345

Figure Lengend Snippet: HaCaT cells were incubated with HPV16 PsVs for 15 min at 4°C, washed, fixed at different time points (as indicated), and stained with anti-CD151 mAb 11G5A and anti-L1 pAb K75 antibodies and analyzed as in . Shown is the ratio of CD151-L1 colocalizing to total L1 pixels. Data (n = 139–163 randomly chosen fields) were analyzed with Wilcoxon rank sum test. For different time points compared to 0:00 hr (control): p=1.44E-04 (0:45 hr), p=3.08E-11 (2:45 hr) and p=2.20E-16 (4:45 hr); difference between 0:45 hr and 2:45 hr: p=6.86E-04, 0:45 hr and 4:45 hr: p=7.12E-08 and 2:45 hr and 4:45 hr: p=0.136. All values are given as mean ± SEM.

Article Snippet: CD151-specific mouse mAb (11G5a) was obtained from Bio-Rad (Munich, Germany).

Techniques: Incubation, Staining

Journal: eLife

Article Title: ADAM17-dependent signaling is required for oncogenic human papillomavirus entry platform assembly

doi: 10.7554/eLife.44345

Figure Lengend Snippet: ( A ) HaCaT cells were incubated for 15 min with HPV16 PsVs, washed, and either directly fixed or after incubation for the indicated times. Then cells were stained with anti-CD151 mAb 11G5A (green) and anti-L1 pAb K75 (red) antibodies and analyzed by superresolution STED microscopy. Representative STED-images for the different time points are shown. From the STED micrographs, we determined the distance of a PsV to its next nearest CD151 cluster and the local intensity of CD151 at the PsV position. ( B ) Increase of the fraction of HPV16 PsVs that are closer than 150 nm to the next CD151 nano-cluster. Statistical significance (n=3) was analyzed with paired t-test: p=0.091 (0:45), p=0.021 (2:45), p=0.017 (4:45), difference between 0:45 and 2:45: p=0.276, between 0:45 and 4:45: p=0.011 and 2:45 and 4:45: p=0.617. Values for every sample were expressed as percentage of the total number of PsVs in the given sample. ( C ) The CD151 intensity at the HPV16 PsV position increases over time. Data (60–65 cells per condition) were analyzed with Wilcoxon rank sum test: p=0.974 (0:45), p=2.53E-08 (4:45), difference between 0:45 and 2:45: p=4.86E-03, between 0:45 and 4:45: p=3.02E-04 and 2:45 and 4:45: p=0.290, and with Welch two sample t-test: p=5.20E-06 (2:45). 15 min time point (0:00) was normalized to 100%. ( D ) HaCaT cells were incubated for 15 min with PsVs, washed, incubated for another 4:45 hr, fixed and incubated with primary antibodies as in (A). Representative images are shown of control siRNA- (upper panel) or ADAM17 siRNA- (lower panel) treated cells. ( E, F ) Quantification of the spatial overlap between CD151 and L1 in HaCaTs ( E ) or in NHEK cells ( F ). The values obtained from the ratio of CD151-L1 colocalizing to total L1 pixels of control siRNA-treated cells (contr. si) were normalized to 100%. Data for HaCaTs (n = 111–129) were analyzed using Wilcoxon rank sum test: p=4.33E-04. NHEKs (n = 180–187); Wilcoxon rank sum test: p=1.75E-05. ( G ) HaCaT control or ADAM17 siRNA-treated cells were incubated with HPV16 PsVs for 5 hr, fixed, and processed for PLA detection using primary antibodies as in (A). Shown is the CD151-L1 PLA signal. ( H ) The ratio of PLA positive signal (red) to nuclear signal (blue) is for contr. si set to 100%. Data (n = 104–115 images) were analyzed using Wilcoxon rank sum test: p=5.77E-10. All values are given as mean ± SEM.

Article Snippet: CD151-specific mouse mAb (11G5a) was obtained from Bio-Rad (Munich, Germany).

Techniques: Incubation, Staining, Microscopy

Journal: eLife

Article Title: ADAM17-dependent signaling is required for oncogenic human papillomavirus entry platform assembly

doi: 10.7554/eLife.44345

Figure Lengend Snippet: For the analysis, five movies were analyzed published in part already in . HeLa cells were tranfected with CD151-CFP (shown in green) and incubated with Alexa Fluor 488 (AF488)-labeled HPV16 pseudovirions (shown in red) for 1.5–4 hr, followed by imaging for 3 min at 0.166 Hz employing TIRF microscopy. ( A ) Overview from the two channels and overlay. The CD151 cluster pattern is not well resolved due to diffraction-limited resolution. ( B ) Magnified views (upper and lower rows are not the same field of view). Images taken from a 180 s sequence show the lateral mobility of a CD151-CFP particle (upper row) and PsV particle (lower row). Spherical CD151 structures often move directional. ( C ) Tracks of the object marked with an arrow in ( B ) and the centered viral particle in ( B ). ( D ) Mean squared displacement (MSD) plotted against time interval (n = 14–15 particles per channel) for CD151-CFP and PsV particles, respectively. ( E ) Diffusion coefficients for CD151-CFP and PsV particles. Statistical significance (n = 14–15 particles per channel) was analyzed with Wilcoxon rank sum test: p=5.89E-03. All values are given as means ± SEM.

Article Snippet: CD151-specific mouse mAb (11G5a) was obtained from Bio-Rad (Munich, Germany).

Techniques: Incubation, Labeling, Imaging, Microscopy, Sequencing, Diffusion-based Assay

Journal: eLife

Article Title: ADAM17-dependent signaling is required for oncogenic human papillomavirus entry platform assembly

doi: 10.7554/eLife.44345

Figure Lengend Snippet: ( A, B ) ADAM17 depletion reduces EGFR-L1 association. ( A ) Representative images of HaCaT cells treated with control or ADAM17-specific siRNA for 48 hr, exposed to HPV16 PsVs for 5 hr, and subsequently stained with EGFR-specific D38B1 (green) and L1-specific 312 F (red) antibodies. ( B ) Quantification of the pixel overlap from the stainings shown in (A). Data for HaCaTs (n = 158–176 randomly chosen fields) were analyzed using Wilcoxon rank sum test: p=5.90E-04. ( C ) EGFR-CD151 association analyzed by fluorescence microscopy. Shown is a deconvoluted image of a HaCaT cells double stained with antibodies specific for EGFR (D38B1) and CD151 (11G5A). EGFR is shown in green, CD151 in red and nuclei in blue. ( D ) EGFR-CD151 overlap analyzed by superresolution microscopy. Representative images of control or ADAM17 depleted HaCaT cells, exposed to HPV16 PsVs for 5 hr, stained with the same primary antibodies as in (C) but CD151 is shown in green and EGFR in red, and analyzed by STED microscopy. ( E ) Pearson’s correlation coefficient (PCC) of staining shown in (D) indicate specific overlap of EGFR and CD151. For the quantification of random overlap, we flipped one image horizontally and vertically to randomize the distribution. Data (n = 60 randomly chosen fields) for each siRNA was compared to control (contr. si) and analyzed using Wilcoxon rank sum test: p<2.20E-16. The difference between contr. and ADAM17 siRNA was not significant (p=0.239). The values for all experiments are given as mean ± SEM.

Article Snippet: CD151-specific mouse mAb (11G5a) was obtained from Bio-Rad (Munich, Germany).

Techniques: Staining, Fluorescence, Microscopy

Journal: eLife

Article Title: ADAM17-dependent signaling is required for oncogenic human papillomavirus entry platform assembly

doi: 10.7554/eLife.44345

Figure Lengend Snippet: HaCaT cells were detached and transfected with CD151-GFP using the Gene pulser Xcell electroporation system (Bio-Rad, Hercules, CA) employing the settings 200 V, 950 μF and 200 Ω. Cells were plated onto poly-Lysine glass-coverslips and after 24 hr membrane sheets were generated applying 100 ms ultrasound sonication pulses at different coverslip locations. The sample was directly fixed using 4% PFA and quenched with 50 mM NH 4 Cl. The sample was blocked with 3% BSA in PBS, washed and imaged in PBS containing 1-(4-tri-methyl-ammonium-phenyl)−6-phenyl-1,3,5-hexatriene p-toluene-sulfonate (TMA-DPH, cat# T-204, Invitrogen) for the visualization of membranes. Imaging was performed using an Olympus IX81 fluorescence microscope essentially as previously described . ( A ) By applying an ultrasound pulse, the apical membrane and cytosolic structures are ripped off leaving behind the basolateral membrane. Intracellular compartments and cytosolic content are removed. Only membrane bound invaginations, cytoskeletal elements and other membrane-anchored structures remain. ( B ) Exemplary image of a plasma membrane sheet. The left panel shows membrane staining by TMA-DPH. The right panel shows overexpressed CD151-GFP. Please note that at the edges double membranes are visible. For this reason, analysis is performed on central areas of the plasma membrane sheets.

Article Snippet: CD151-specific mouse mAb (11G5a) was obtained from Bio-Rad (Munich, Germany).

Techniques: Transfection, Electroporation, Generated, Sonication, Imaging, Fluorescence, Microscopy, Staining

Journal: eLife

Article Title: ADAM17-dependent signaling is required for oncogenic human papillomavirus entry platform assembly

doi: 10.7554/eLife.44345

Figure Lengend Snippet: ( A ) Representative images of membrane sheets generated from HaCaT cells stained for CD151 with 11G5A mAb (green) and HPV16 L1 with K75 pAb (red). Shown are membrane sheets generated from control siRNA (upper and middle row) and ADAM17 siRNA (lower row) transfected cells that were incubated for 5 hr without (upper row) or with PsVs (middle and lower row). ( B ) HPV16 treatment diminishes CD151 cluster size, an effect that is abolished upon ADAM17 depletion. ADAM17+/PsVs + was compared to the two other conditions and analyzed with Wilcoxon rank sum test: p=3.10E-04 (for ADAM17+/PsVs-) and p=7.01E-09 (for ADAM17-/PsVs+). ( C ) HPV16 treatment diminishes the CD151 surface level, an effect that is abolished upon ADAM17 depletion. Statistical analysis was performed comparing to the condition ADAM17+/PsVs + and analyzed with Welch two sample t-test: p=4.16E-05 (for ADAM17+/PsVs-) and two sample t-test: p=2.21E-07 (for ADAM17-/PsVs+). For ( B ) and ( C ) (n = 60), membrane sheets for each condition were collected from three biological replicates. Values are given as mean ± SEM. The condition ADAM17+/PsVs- was set to 100%.

Article Snippet: CD151-specific mouse mAb (11G5a) was obtained from Bio-Rad (Munich, Germany).

Techniques: Generated, Staining, Transfection, Incubation

Journal: eLife

Article Title: ADAM17-dependent signaling is required for oncogenic human papillomavirus entry platform assembly

doi: 10.7554/eLife.44345

Figure Lengend Snippet: ( A ) EGFR blocking-antibody Cetuximab decreases HPV16 PsVs infection rate. HaCaT cells were pre-incubated with stated concentrations (µg/ml) of Cetuximab for 1 hr, infected with HPV16 PsVs and 24 hr later luciferase counts were measured. Samples (n = 8) were analyzed using two sample t-test: p=3.67E-08 (1 µg/ml), and Welch two sample t-test: p=4.42E-06 (10 µg/ml) and p=6.76E-07 (30 µg/ml). Infection rate is given as mean ± SEM and the mean for control-treated cells (contr.) was set to 100%. ( B ) HaCaT cells were depleted of ADAM17 for 48 hr. Next, cells were starved for 1 hr in medium without FCS, either left non-treated or treated with EGF (20 ng/ml) for 5 min, and incubated with HPV16 PsVs for 5 min. Western blots show phosphorylated and total ERK1 and 2 for the indicated conditions; α-tubulin was used as a loading control. ( C ) The amount of phosphorylated ERK1 (black) and ERK2 (gray) is shown as a ratio of phosphorylated to total ERK form. Data for ERK1 (n = 8–9) compared to the corresponding ERK1 of the contr. si were analyzed with two sample t-test: p=3.85E-08 (ADAM17 si) and Welch two sample t-test: p=1.73E-05 (ADAM17 si + EGF), and the difference between ADAM17 si and ADAM17 si + EGF: p=3.15E-06. Data for ERK2 (n = 9) when compared to the corresponding ERK2 of control siRNA-treated cells (contr. si); Welch two sample t-test: p=1.61E-06 (ADAM17 si) and p=1.13E-03 (ADAM17 si + EGF), and the difference between ADAM17 si and ADAM17 si + EGF: p=2.62E-05. The values are given as mean ± SEM and the mean for contr. si was set to 100%. ( D ) Infection assay after EGF reconstitution. HaCaT cells were transfected with control (left panel) or ADAM17 siRNA (right panel) for 48 hr, left non-treated or treated with EGF (20 ng/ml) and infected with HPV16 PsVs. Samples shown on the left panel (n = 7) were analyzed using Wilcoxon rank sum test: p=2.33E-03 and on the right panel (n = 9) with two sample t-test: p=1.30E-06. Infection rate (in %) is given as mean ± SEM and the mean of EGF-non-treated control siRNA-treated cells was normalized to 100%. Because of simplicity, the data is shown in separate graphs (panels). ( E ) Shown is specific CD151-L1 PLA signal (red). HaCaT cells were depleted of ADAM17 and either left non-treated, or pre-treated with EGF (20 ng/ml) for 5 min and afterwards incubated with PsVs for 5 hr. ( F ) The ratio of PLA-positive signal (red) to nuclear signal (blue) (in %) is given as mean ± SEM. and the mean was normalized to EGF-non-treated control cells (shown in ). Data (n = 128–152 images) were analyzed using Wilcoxon rank sum test: p=7.97E-06.

Article Snippet: CD151-specific mouse mAb (11G5a) was obtained from Bio-Rad (Munich, Germany).

Techniques: Blocking Assay, Infection, Incubation, Luciferase, Western Blot, Transfection

Journal: eLife

Article Title: ADAM17-dependent signaling is required for oncogenic human papillomavirus entry platform assembly

doi: 10.7554/eLife.44345

Figure Lengend Snippet:

Article Snippet: CD151-specific mouse mAb (11G5a) was obtained from Bio-Rad (Munich, Germany).

Techniques: Flow Cytometry, Recombinant, Sequencing, Integrity Assay, Enzyme-linked Immunosorbent Assay, Luciferase, Software