Journal: Cell reports

Article Title: TBC1D5-Catalyzed Cycling of Rab7 Is Required for Retromer-Mediated Human Papillomavirus Trafficking during Virus Entry

doi: 10.1016/j.celrep.2020.107750

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Rabbit monoclonal antibody anti-Rab7 (D95F2) , Cell Signaling Technology , Cat#9367 RRID: AB_1904103.

Techniques: Produced, Virus, Recombinant, Electron Microscopy, Modification, Saline, Protein Extraction, In Situ, Cloning, Transfection, Bicinchoninic Acid Protein Assay, Staining, Control, shRNA, Mutagenesis, Software, Flow Cytometry, Microscopy, Molecular Weight

Journal: Traffic (Copenhagen, Denmark)

Article Title: Rab18 inhibits secretory activity in neuroendocrine cells by interacting with secretory granules.

doi: 10.1111/j.1600-0854.2007.00570.x

Figure Lengend Snippet: Figure 2: Colocalization of GFP-Rab18 recombinant protein with markers of different intracellular compartments in transiently transfected PC12 cells. Colocalization analysis of GFP-Rab18 (green) with markers for different intracellular compartments (red), including early endosomes (EEA1), late endosomes (Rab7) and ER-to-Golgi, COPII-coated vesicles (SEC23), in GFP-Rab18-expressing PC12 cells. For all markers, no significant overlapping was observed between fluorescent signals either under basal (upper panels) or Kþ-stimulated conditions (lower panels). Scale bars, 5 mm.

Article Snippet: Anti-human SEC23 and anti-human Rab7 were from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

Techniques: Recombinant, Transfection, Expressing

Journal: Brain

Article Title: SLC30A9 mutation affecting intracellular zinc homeostasis causes a novel cerebro-renal syndrome

doi: 10.1093/brain/awx013

Figure Lengend Snippet: SLC30A9 protein subcellular localization, effect on Wnt signaling and structural modeling. ( A ) Overexpression of SLC30A9 protein fused to enhanced green fluorescent protein (EGFP) and immunofluorescent staining of different endosomal, Golgi and ER markers (CD63, EEA1, Rab7, GM130 and Calnexin) showing partial co-localization with endoplasmic reticulum in SH-SY5Y cells. On the bottom left side of the merged image of Calnexin marker panel is a magnification of the dashed frame, showing the co-localization in a single cell. Scale bars = 10 µm. ( B ) TOP flash reporter assay of neuroblastoma (SH-SY5Y) cells showing enhanced Wnt signalling in both SLC30A9 wild-type and mutant transfected cells. There was no significant difference between wild-type and mutant transfected cells. ( C ) Structural model of ZnT-9 protein showing the p.(A350del) in the fourth transmembrane helix, putatively causing destabilization of the protein structure in general, affecting the TM metal binding site specifically.

Article Snippet: Cells were then incubated with primary polyclonal goat anti-human EEA1 (Sc-6414; Santa Cruz Biotechnology), polyclonal rabbit anti- human Rab7 (Sc-10767; Santa Cruz Biotechnology), mouse monoclonal anti- human CD63 (CBL553; Merck Millipore) or polyclonal rabbit anti-human Calnexin (Sc-11397; Santa Cruz Biotechnology) antibodies for 1 h. Post-incubation, cells were washed twice with PBST and incubated with secondary donkey anti-goat IgG Alexa Fluor® 546 (A-11056; Invitrogen), goat anti-rabbit IgG Alexa Fluor® 546 (A-11010; Invitrogen) or donkey anti-mouse IgG Alexa Fluor® 546 (A-10036; Invitrogen) accordingly for 1 h. Cells were then washed twice with 1 × PBST and mounted using Vectashield containing DAPI (H-1200; Vector Laboratories).

Techniques: Over Expression, Staining, Marker, Reporter Assay, Mutagenesis, Transfection, Binding Assay

Journal: Cells

Article Title: AMPK Activation Downregulates TXNIP, Rab5, and Rab7 Within Minutes, Thereby Inhibiting the Endocytosis-Mediated Entry of Human Pathogenic Viruses

doi: 10.3390/cells14050334

Figure Lengend Snippet: AMPK activation does not influence the surface expression of CD155 or the LDL receptor, but it reduces cellular TXNIP, Rab5, and Rab7 and inhibits viral endocytosis. ( A ) FACS analyses of the CD155 and LDLR expression at the plasma membrane. FMO: control without the first antibodies. ( B ) Western blotting analysis of TXNIP, Rab5, and Rab7 after 24 h and for TXNIP after 2 h. ( C ) Confocal microscopy of TXNIP expression after 2 h. blue: DAPI; magenta: TXNIP. Scale bars 20 µm. ( D ) YFV and ( E ) SARS-CoV-2 replication in 293T cells treated with ASA SA and transfected with a constitutive active NF-κB. ( F ) Analyses of proteasomal proteases with the compounds. Bortezomib served as inhibitor control.

Article Snippet: Antibodies: Anti-PVR (CD155) Antibody, clone 4B3, ZooMab ® Rabbit Monoclonal recombinant, Sigma Aldrich; anti-LDL Receptor Antibody, clone 2N19, ZooMab ® Rabbit Monoclonal recombinant, Sigma Aldrich (Taufkirchen, Germany); anti-Rab5A Antibody #2143, Cell Signaling; anti-Rab7 (D95F2) XP ® Rabbit mAb #9367, Cell Signaling; and anti-TXNIP Antibody, clone 1K14 ZooMAb ® Rabbit Monoclonal, Sigma Aldrich; anti-β-Actin Antibody, clone 6L12, ZooMAb ® Rabbit Monoclonal, Sigma Aldrich; anti-SARS-CoV-2 Spike Protein (S1-NTD) Antibody #56996, Cell Signaling, anti-SARS-CoV/SARS-CoV-2 NSP8 Monoclonal Antibody (5A10), Invitrogen; and anti-LAMP1 (D4O1S) Mouse mAb #15665, Cell Signaling; anti-GRP78 Polyclonal Antibody, Invitrogen.

Techniques: Activation Assay, Expressing, Clinical Proteomics, Membrane, Control, Western Blot, Confocal Microscopy, Transfection

Journal: Cells

Article Title: AMPK Activation Downregulates TXNIP, Rab5, and Rab7 Within Minutes, Thereby Inhibiting the Endocytosis-Mediated Entry of Human Pathogenic Viruses

doi: 10.3390/cells14050334

Figure Lengend Snippet: AMPK activation downregulates Rab5, Rab7, and TXNIP rapidly. Confocal microscopy of Rab5 (magenta), Rab7 (magenta), and TXNIP (magenta) after SA ( A , C ), AMPK ( A ), ULK activator ( A ), and cycloheximide ( B ) treatment. ( C ) Analyses of the time-dependent downregulation of Rab5, Rab7, and TXNIP. Scale bars: 20 µm.

Article Snippet: Antibodies: Anti-PVR (CD155) Antibody, clone 4B3, ZooMab ® Rabbit Monoclonal recombinant, Sigma Aldrich; anti-LDL Receptor Antibody, clone 2N19, ZooMab ® Rabbit Monoclonal recombinant, Sigma Aldrich (Taufkirchen, Germany); anti-Rab5A Antibody #2143, Cell Signaling; anti-Rab7 (D95F2) XP ® Rabbit mAb #9367, Cell Signaling; and anti-TXNIP Antibody, clone 1K14 ZooMAb ® Rabbit Monoclonal, Sigma Aldrich; anti-β-Actin Antibody, clone 6L12, ZooMAb ® Rabbit Monoclonal, Sigma Aldrich; anti-SARS-CoV-2 Spike Protein (S1-NTD) Antibody #56996, Cell Signaling, anti-SARS-CoV/SARS-CoV-2 NSP8 Monoclonal Antibody (5A10), Invitrogen; and anti-LAMP1 (D4O1S) Mouse mAb #15665, Cell Signaling; anti-GRP78 Polyclonal Antibody, Invitrogen.

Techniques: Activation Assay, Confocal Microscopy

Journal: Cellular microbiology

Article Title: The fungal pathogen Cryptococcus neoformans manipulates macrophage phagosome maturation.

doi: 10.1111/cmi.12394

Figure Lengend Snippet: Fig. 1. Acquisition of Rab GTPases onto the Cryptococcus-containing phagosome. At 15 min and 2 h, Rab5 (A), Rab11 (B), Rab9 (C) and Rab7 (D) recruitment to phagosomes containing live cryptococci was monitored. Recruitment at 5 min is comparable between phagosomes containing live C. neoformans H99, heat-killed H99 and a variety of inert targets (E). All data were collected from immunofluorescence analysis of J774 phagocytosed particles. All bars represent data collected from observing 100–664 phagosomes for each target at each time point over three to six biological repeats, mean ± SEM. Data presented for H99 and HK H99 at 15 and 120 min are replicated in A and E. ***P < 0.001, **P < 0.01 Fisher’s exact test.

Article Snippet: Cells were then treated with 50 nM NH4Cl for 10 min and permeabilized in 0.1% Triton X-100 for 4 min. Coverslips were then blocked with 5% goat serum for 1 h and then washed in PBS before being treated with 0.5 μg ml−1 primary antibody (monoclonal rabbit anti-Rab5, Rab7, Rab9 or Rab11) (Cell Signaling) with 2.5 μg ml−1 human IgG for 30 min. After PBS washing, coverslips were then treated with 2 μg ml−1 secondary goat anti-rabbit IgGFITC (Sigma) with 10 μg ml−1 human IgG for 30 min. Coverslips were visualized with a Nikon Eclipse Ti-S microscope, Plan Apo 60×/1.40 NA oil DIC objective (Nikon) and captured with QICAM Fast1394 camera (QImaging).

Techniques: