Journal: bioRxiv

Article Title: SORLA-driven endosomal trafficking regulates the oncogenic fitness of HER2

doi: 10.1101/299586

Figure Lengend Snippet: SORLA is highly expressed in HER2 breast cancer cells and associates and co-traffics with HER2 ( a ) Western blot analysis of SORLA and HER2 in breast cancer cell lines. α-tubulin is a loading control. ( b ) Confocal microscopy imaging of HER2 (magenta) and EEA-1 (green) in BT474, MDA-MB-361 and JIMT-1 cells. ( c ) Endogenous SORLA (green), HER2 (magenta) and EEA-1 (white) staining in MDA-MB-361 cells (top panel). Endogenous HER2 (magenta) and VPS35 (white) staining in JIMT-1 cells expressing SORLA-GFP (green) (bottom panel). ( d ) Live-cell TIRF imaging of AlexaFluor-568-labeled trastuzumab (Tz-568; red) and SORLA-GFP (green) in MDA-MB-361 cells. ( e ) Co-immunoprecipitation of endogenous SORLA with endogenous HER2 in MDA-MB-361 and BT474 cells. ( f ) Schematic of the SORLA protein domains and summary of the constructs used. ( g ) Co-immunoprecipitation of endogenous HER2 with different SORLA-GFP fragments in MDA-MB-361 cells. Where immunoblots and micrographs are shown, these are representative of n = 3 independent experiments; IB: immunoblotting, IP: Immunoprecipitation. ECD: extracellular domain; TM: transmembrane domain; CD: cytosolic domain.

Article Snippet: Stainings were performed using antibodies against HER2 (Herceptin (Roche) 0.15 μg/ml or mouse monoclonal antibody (ThermoScientific; MA5-14057; dilution of 1:300), LAMP1 (Santa Cruz; SC-20011 (H4A3), dilution 1:50), SORLA (rabbit monoclonal, CM Petersen Lab, Århus University, dilution of 1:300), EEA-1 (goat polyclonal, Santa Cruz; sc-6415, dilution 1:50), VPS35 (goat polyclonal, Abcam; ab10099, dilution 1:300), CD63 (mouse mAb, Hybridoma Bank; H5C6, dilution 1:300) and Rab11 (rabbit polyclonal, Cell Signaling Technology, #5589, dilution 1:100).

Techniques: Western Blot, Confocal Microscopy, Imaging, Staining, Expressing, Labeling, Immunoprecipitation, Construct

Journal: Frontiers in Cellular and Infection Microbiology

Article Title: Insights into the function of ESCRT complex and LBPA in ASFV infection

doi: 10.3389/fcimb.2023.1163569

Figure Lengend Snippet: VPS4A and vesicle reorganization at viral factories. Representative confocal images of empty and shVPS4A Vero cells uninfected (MOCK) or infected with Ba71V at a MOI of 1 pfu/cell for 16 (h) After fixation, cells were stained for viral core protein p150 (red), DNA (TO-PRO-3, blue), and vesicle membrane marker EEA1 (green) (A) , CD63 (green) (B) , or Lamp1 (green) (C) . Scale bar: 20 µm.

Article Snippet: Mouse monoclonal antibodies used were as follows: ALIX (Q19) (sc-49268), EEA1 (BD Biosciences, 610457), CD63 (H5C6) (Novus Biologicals, NBP2-42225), p150 (Ingenasa, 17AH2), p72 (Ingenasa,1BC11), p72 (Ingenasa, 18BG3), p30 (a gift from J.M.

Techniques: Infection, Staining, Membrane, Marker

Journal: Frontiers in Microbiology

Article Title: Induction of Tyrosine Phosphorylation of UV-Activated EGFR by the Beta-Human Papillomavirus Type 8 E6 Leads to Papillomatosis

doi: 10.3389/fmicb.2017.02197

Figure Lengend Snippet: HPV8E6 alters the intracellular trafficking of the EGFR after UV-irradiation. (A) FACS analysis of fixed and non-permeabilized pLXSN-empty vector or HPV8E6-NTERT and HaCat cells. Cells were left untreated or irradiated with a dose of UV-light of 40 mJ/cm 2 and harvested 30 or 75 min after UV-exposure. The cells were stained with the anti-EGFR-AF488 or the isotype control IgG-AF488 coupled antibody. The graphs represent the overlays of the EGFR-positive non-irradiated cells (blue line) with those harvested 30 min or 75 min post UV (red line), respectively. (The light and dark gray graphs represent the cells stained with the isotype control. (B) Immunofluorescence analysis. N/TERT harboring the pLXSN or pLXSN-HPV8E6 were fixed and permeabilized after UV-exposure, as indicated. Immunofluorescence was performed with anti-EGFR-AF488 (green) and with the anti-EEA1 (red, upper part) or the anti-CD63 (red, lower part) antibody. Nuclei were stained with DAPI. The photos represent representative images, respectively. A more detailed section in a higher magnification is provided including the single stains. The scale bars are given at the bottom (magnification 1:1000). (C) Quantification of the number of EGFR positive early endosomes (EGFR and EEA1 positive), and of the CD63 and EGFR positive ILVs (in each case indicated by yellow spots). Bar graphs represent the fold changes of the number of yellow spots per cell, indicative for a co-localization of EGFR and EEA1 or CD63 15, 30, and 75 min after UV-irradiation. The values were obtained by counting the yellow spots in a total of 70 to 130 cells, obtained from two independent experiments. Error bars indicate the standard deviations, the asterisks indicate the significance ( ∗∗ p < 0.001, ∗ p = 0.052)

Article Snippet: The anti-EEA1 (BD Clone 14) and CD63 (BD clone H5C6) were purchased from BD Biosciences and M2-FLAG-affinity Gel and antibodies from Sigma and the GFP-tag antibody from Thermo-Scientific (A-11122).

Techniques: Irradiation, Plasmid Preparation, Staining, Immunofluorescence

Journal: PLoS ONE

Article Title: Antiviral Role of IFITM Proteins in African Swine Fever Virus Infection

doi: 10.1371/journal.pone.0154366

Figure Lengend Snippet: (A). Confocal microscopy images of Vero-IFITM cells or controls containing the empty vector stained with endosomal markers EEA1 (EE), CD63 (MVB), Rab7 (LE) and Lamp1 (LY). Endosomes were predominantly dispersed in the cytoplasm of cells containing the empty vector or concentrated to the perinuclear area in Vero-IFITM2 and 3 cells. (B). The change in distribution was quantified by measuring the mean distance to the nucleus of the different endosomal markers in x, y and z planes as described in materials and methods section. As shown in graphics, distance was reduced in Vero-IFITM2 and 3 cells. Graphics depict mean±SD of N = 30 cells per condition. Statistical significance was evaluated by a one-way ANOVA followed by Bonferroni’s multiple comparison test. Differences are marked with asterisks as indicated (* p <0.05; ** p <0.01).

Article Snippet: Escribano, INIA); ASFV mouse monoclonal antibodies anti-p72 (clone 1BC11 for immunofluorescence 1:1,000 or clone 18BG3 for WB 1:2,000) and anti-p150 (clone 17AH2, Ingenasa), 1:1,000; mouse monoclonal to CD63 (Developmental Studies Hybridoma Bank, University of Iowa, clone H5C6), 1:200; rabbit polyclonal to Lamp1 (Abcam), 1:50; rabbit polyclonal to EEA1 and Rab7 (Cell Signalling), 1:50.

Techniques: Confocal Microscopy, Plasmid Preparation, Staining, Comparison