Journal:

Article Title: Neutralizing Antibodies Inhibit Axonal Spread of Herpes Simplex Virus Type 1 to Epidermal Cells In Vitro

doi:

Figure Lengend Snippet: Confocal micrographs of HSV-infected neurons stained for gC antigen at 15 hpi and after addition of human anti-gD monoclonal antibody (top) or control medium (bottom). The HSV inoculum (5 TCID50/cell) was aspirated after 1 h of incubation, and the cells were carefully washed once with HBSS. The HSV-infected or mock-infected dissociated neuronal cultures, incubated with a 1:2,500 dilution (400 ng/ml) of human anti-gD antibody, were fixed in 2.5% formaldehyde (ProSci Tech) in Sorensons buffer (pH 7.4) for 30 min and permeabilized with 0.1% Triton X-100 (Sigma) in PBS for 20 min. Nonspecific staining was blocked by incubation with 5% mouse serum in HBSS for 15 min. The cells on coverslips were then incubated with fluorescein isothiocyanate-conjugated anti-gC1 antibody (Syva Microtrak) (1:100 dilution), rinsed three times with HBSS, and mounted in mounting fluid (Syva Microtrak). Stained neurons were examined with a Bio-Rad MRC 600 confocal microscope. Note the similar distributions of gC antigen in the axon and cytoplasm in both micrographs. Bars, 40 μm (top) and 20 μm (bottom).

Article Snippet: The HSV-infected or mock-infected dissociated neuronal cultures, incubated with a 1:2,500 dilution (400 ng/ml) of human anti-gD antibody, were fixed in 2.5% formaldehyde (ProSci Tech) in Sorensons buffer (pH 7.4) for 30 min and permeabilized with 0.1% Triton X-100 (Sigma) in PBS for 20 min. Nonspecific staining was blocked by incubation with 5% mouse serum in HBSS for 15 min.

Techniques: Infection, Staining, Incubation, Microscopy

Journal: Scientific Reports

Article Title: UBXD1 is a mitochondrial recruitment factor for p97/VCP and promotes mitophagy

doi: 10.1038/s41598-018-30963-z

Figure Lengend Snippet: UBXD1 translocates to depolarized mitochondria in a Parkin-dependent manner. ( A ) HeLa cells transfected with expression constructs for FLAG-UBXD1, mitoYFP, and Parkin were treated with 10, 15, 25, 40, or 50 µM CCCP for 6 hours or left untreated as control. Cells were fixed, stained using mouse anti-FLAG antibodies to detect UBXD1 and analyzed by confocal microscopy. ( B ) HeLa cells transfected with expression constructs for FLAG-UBXD1, mitoYFP, and Parkin or vector control were treated with 50 µM CCCP for six hours of left untreated as controls. Cells were analyzed as above.

Article Snippet: To detect UBXD1 protein, protein lysates of HeLa cells were prepared using RIPA buffer, separated on 12%SDS-PAGE, and analyzed using mouse anti-UBXD1 (Abcam, 80659), mouse α-GAPDH (1:3000; Santa Cruz #sc-32233), anti-FLAG (1:1000, Sigma F1804), anti-myc (1:2000, Sigma M5546) and anti-mouse HRP (1:20000; ThermoFisher) antibodies.

Techniques: Transfection, Expressing, Construct, Control, Staining, Confocal Microscopy, Plasmid Preparation

Journal: Scientific Reports

Article Title: UBXD1 is a mitochondrial recruitment factor for p97/VCP and promotes mitophagy

doi: 10.1038/s41598-018-30963-z

Figure Lengend Snippet: Mitochondrial translocation of UBXD1 in HEK293 cells and during Parkin-independent mitophagy. ( A ) HEK293 cells transfected with expression constructs for FLAG-UBXD1 and mitoYFP were treated for 6 hours with 50 µM CCCP or left untreated as controls. After fixation and anti-FLAG staining, cells were analyzed by confocal microscopy. Fluorescence intensities of FLAG-UBXD1 (red) and mitoYFP (green) along the white line are plotted. Shown are representative images of three independent experiments. ( B ) HeLa cells transfected with expression constructs for FLAG-UBXD1 and mitoYFP were treated for 24 hours with 1 mM DFP or left untreated as control. Cells were fixed, stained using mouse anti-FLAG antibodies to detect UBXD1 and analyzed by confocal microscopy. ( C ) HeLa cells transfected with expression constructs for GFP-LC3 and mitoDsRed were treated for 24 hours with 1 mM DFP, treated for 6 hours with 100 nM bafilomycin or left untreated, fixed and analyzed by confocal microscopy.

Article Snippet: To detect UBXD1 protein, protein lysates of HeLa cells were prepared using RIPA buffer, separated on 12%SDS-PAGE, and analyzed using mouse anti-UBXD1 (Abcam, 80659), mouse α-GAPDH (1:3000; Santa Cruz #sc-32233), anti-FLAG (1:1000, Sigma F1804), anti-myc (1:2000, Sigma M5546) and anti-mouse HRP (1:20000; ThermoFisher) antibodies.

Techniques: Translocation Assay, Transfection, Expressing, Construct, Staining, Confocal Microscopy, Fluorescence, Control

Journal: Scientific Reports

Article Title: UBXD1 is a mitochondrial recruitment factor for p97/VCP and promotes mitophagy

doi: 10.1038/s41598-018-30963-z

Figure Lengend Snippet: VIM and PUB, but not the UBX domain of UBXD1 are dispensable for mitochondrial translocation during mitophagy. ( A ) HeLa cells transfected with mitoYFP-T2A-Parkin-myc3 and FLAG-UBXD1, FLAG-UBXD1ΔPUB, FLAG-UBXD1ΔVIM, FLAG-UBXD1ΔUBX, or FLAG-UBXonly expression constructs were treated with CCCP for 6 hours or left untreated as control, stained using mouse anti-FLAG antibodies and analyzed by confocal microscopy. ( B ) The ratio of mitochondrial to total FLAG-UBXD1 or various FLAG-UBXD1 variants as measure for mitochondrial translocation was quantified by image analysis of confocal pictures obtain from cells treated as in A. Shown are box plots of three independent experiments with at least 15 cells per experiment and condition. Statistical significance was assessed by ANOVA followed by Student’s t-test using Bonferroni correction to account for multiple comparisons. *** denotes p-values < 0.001, n.s. – no significant difference.

Article Snippet: To detect UBXD1 protein, protein lysates of HeLa cells were prepared using RIPA buffer, separated on 12%SDS-PAGE, and analyzed using mouse anti-UBXD1 (Abcam, 80659), mouse α-GAPDH (1:3000; Santa Cruz #sc-32233), anti-FLAG (1:1000, Sigma F1804), anti-myc (1:2000, Sigma M5546) and anti-mouse HRP (1:20000; ThermoFisher) antibodies.

Techniques: Translocation Assay, Transfection, Expressing, Construct, Control, Staining, Confocal Microscopy

Journal: Scientific Reports

Article Title: UBXD1 is a mitochondrial recruitment factor for p97/VCP and promotes mitophagy

doi: 10.1038/s41598-018-30963-z

Figure Lengend Snippet: UBXD1 recruits p97 to mitochondria under mitophagic conditions. ( A ) HeLa cells transfected with expression plasmids for FLAG-UBXD1 and mitoYFP-T2A-Parkin-myc3 were treated with CCCP for 6 hours or left untreated as control. Cells were fixed, stained using rabbit anti-FLAG and mouse anti-p97 antibodies and analyzed by confocal microscopy. ( B ) HeLa cells transfected with expression plasmids for FLAG-UBXD1 or vector control and mitoYFP-T2A-Parkin-myc3 were treated as in A. To quantify p97 redistribution to mitochondria, the ratio of mitochondrial p97 to total p97 was determined by image analysis of confocal images. Shown are box plots of three independent experiments with at least 15 cells per experiment and condition. Statistical significance was assessed by ANOVA followed by Student’s t-test using Bonferroni correction to account for multiple comparisons. ***Denotes p-values < 0.001.

Article Snippet: To detect UBXD1 protein, protein lysates of HeLa cells were prepared using RIPA buffer, separated on 12%SDS-PAGE, and analyzed using mouse anti-UBXD1 (Abcam, 80659), mouse α-GAPDH (1:3000; Santa Cruz #sc-32233), anti-FLAG (1:1000, Sigma F1804), anti-myc (1:2000, Sigma M5546) and anti-mouse HRP (1:20000; ThermoFisher) antibodies.

Techniques: Transfection, Expressing, Control, Staining, Confocal Microscopy, Plasmid Preparation

Journal: Scientific Reports

Article Title: UBXD1 is a mitochondrial recruitment factor for p97/VCP and promotes mitophagy

doi: 10.1038/s41598-018-30963-z

Figure Lengend Snippet: The UBX domain of UBXD1 is essential for mitochondrial translocation of p97. HeLa cells transfected with expression plasmids for FLAG-UBXD1 or variants of FLAG-UBXD1 and mitoYFP-T2A-Parkin-myc3 were treated with CCCP for 6 hours or left untreated as control. Fixed cells were stained using rabbit anti-FLAG and mouse anti-p97 antibodies and analyzed by confocal microscopy. The box plots represent three independent experiments with at least 15 cells/experiment/condition. Statistical significance was assessed by ANOVA followed by Student’s t-test using Bonferroni correction to account for multiple comparisons. *denotes p-values < 0.05, ***p-values < 0.001, n.s. – no significant difference.

Article Snippet: To detect UBXD1 protein, protein lysates of HeLa cells were prepared using RIPA buffer, separated on 12%SDS-PAGE, and analyzed using mouse anti-UBXD1 (Abcam, 80659), mouse α-GAPDH (1:3000; Santa Cruz #sc-32233), anti-FLAG (1:1000, Sigma F1804), anti-myc (1:2000, Sigma M5546) and anti-mouse HRP (1:20000; ThermoFisher) antibodies.

Techniques: Translocation Assay, Transfection, Expressing, Control, Staining, Confocal Microscopy

Journal: Scientific Reports

Article Title: UBXD1 is a mitochondrial recruitment factor for p97/VCP and promotes mitophagy

doi: 10.1038/s41598-018-30963-z

Figure Lengend Snippet: The VIM domain of UBXD1 is not involved in mitochondrial translocation and p97 recruitment. HeLa cells transfected with expression plasmid for FLAG-VIMonly and mitoYFP-T2A-Parkin-myc3 were treated with CCCP for 6 hours or left untreated as control. Fixed cells were stained using rabbit anti-FLAG and mouse anti-p97 antibodies and analyzed by confocal microscopy. The box plots represent three independent experiments with at least 15 cells/experiment/condition. Statistical significance was assessed by ANOVA followed by Student’s t-test using Bonferroni correction to account for multiple comparisons. n.s. – no significant difference.

Article Snippet: To detect UBXD1 protein, protein lysates of HeLa cells were prepared using RIPA buffer, separated on 12%SDS-PAGE, and analyzed using mouse anti-UBXD1 (Abcam, 80659), mouse α-GAPDH (1:3000; Santa Cruz #sc-32233), anti-FLAG (1:1000, Sigma F1804), anti-myc (1:2000, Sigma M5546) and anti-mouse HRP (1:20000; ThermoFisher) antibodies.

Techniques: Translocation Assay, Transfection, Expressing, Plasmid Preparation, Control, Staining, Confocal Microscopy

Journal: Scientific Reports

Article Title: UBXD1 is a mitochondrial recruitment factor for p97/VCP and promotes mitophagy

doi: 10.1038/s41598-018-30963-z

Figure Lengend Snippet: HeLa cells transfected with expression plasmids for mitochondria-targeted dsRED (mitodsRED) and YFP-UBXD1 or YFP fused to the mitochondrial membrane targeting signal ActA were fixed, stained using mouse anti-p97 antibodies, and analyzed by confocal microscopy. Shown are representative images out of three independent experiments. The box plots represent three independent experiments with at least 15 cells/experiment/condition. Statistical significance was assessed by by Student’s t-test. ***denotes p < 0.001, n.s. – no significant difference.

Article Snippet: To detect UBXD1 protein, protein lysates of HeLa cells were prepared using RIPA buffer, separated on 12%SDS-PAGE, and analyzed using mouse anti-UBXD1 (Abcam, 80659), mouse α-GAPDH (1:3000; Santa Cruz #sc-32233), anti-FLAG (1:1000, Sigma F1804), anti-myc (1:2000, Sigma M5546) and anti-mouse HRP (1:20000; ThermoFisher) antibodies.

Techniques: Transfection, Expressing, Membrane, Staining, Confocal Microscopy

Journal: Scientific Reports

Article Title: UBXD1 is a mitochondrial recruitment factor for p97/VCP and promotes mitophagy

doi: 10.1038/s41598-018-30963-z

Figure Lengend Snippet: UBXD1 promotes mitophagy. ( A ) HeLa cells transfected with expression plasmids for FLAG-UBXD1 or vector control, mcherry-Parkin and GFP-LC3 were fixed, stained using mouse anti-FLAG antibodies, and analyzed by confocal microscopy. ( B ) HeLa cells transfected with expression plasmids for UBXD1 or vector control and mKeima-T2A-Parkin-myc3 were treated for 12 hours with CCCP or left untreated and analyzed by flow cytometry. Shown are representative density plots (left panels). The box plot represents 6 independent experiments with in total 11 technical replicates. Statistical significance was assessed by ANOVA followed by Student’s t-test using Bonferroni correction to account for multiple comparisons. *** marks p-values < 0.001, n.s. – no significant difference.

Article Snippet: To detect UBXD1 protein, protein lysates of HeLa cells were prepared using RIPA buffer, separated on 12%SDS-PAGE, and analyzed using mouse anti-UBXD1 (Abcam, 80659), mouse α-GAPDH (1:3000; Santa Cruz #sc-32233), anti-FLAG (1:1000, Sigma F1804), anti-myc (1:2000, Sigma M5546) and anti-mouse HRP (1:20000; ThermoFisher) antibodies.

Techniques: Transfection, Expressing, Plasmid Preparation, Control, Staining, Confocal Microscopy, Flow Cytometry

Journal: Scientific Reports

Article Title: UBXD1 is a mitochondrial recruitment factor for p97/VCP and promotes mitophagy

doi: 10.1038/s41598-018-30963-z

Figure Lengend Snippet: UBXD1 levels influence mitophagic flux. ( A ) Using CRISPR/Cas9, UBXD1 was targeted in HeLa cells and several alleles of UBXD1 were replaced with a reporter cassette coding for secreted Gaussia luciferase. Shown are schematics of CRISPR/Cas9 strategy, ( B ) PCR analysis of reporter integration, ( C ) and levels of UBXD1 in HeLa and HeLa UBXD1-low detected using mouse anti-UBXD1 antibodies. ( D ) HeLa cells HeLa UBXD1-low transfected with mKeima-T2A-Parkin-myc3 were treated for 12 hours with 10, 25 or 50 µM CCCP or left untreated as controls and were analyzed by flow cytometry. The box plot represents 5 independent experiments. ( E ) Shown are representative density plots of the analysis shown in D. Statistical significance was assessed by ANOVA followed by Student’s t-test using correction to account for multiple comparisons according to Holm. * marks p-value < 0.05, **p-value < 0.01, n.s. – no significant difference.

Article Snippet: To detect UBXD1 protein, protein lysates of HeLa cells were prepared using RIPA buffer, separated on 12%SDS-PAGE, and analyzed using mouse anti-UBXD1 (Abcam, 80659), mouse α-GAPDH (1:3000; Santa Cruz #sc-32233), anti-FLAG (1:1000, Sigma F1804), anti-myc (1:2000, Sigma M5546) and anti-mouse HRP (1:20000; ThermoFisher) antibodies.

Techniques: CRISPR, Luciferase, Transfection, Flow Cytometry

Journal: Journal of Cellular and Molecular Medicine

Article Title: Nitric oxide and P-glycoprotein modulate the phagocytosis of colon cancer cells

doi: 10.1111/j.1582-4934.2010.01137.x

Figure Lengend Snippet: Effect of nitric oxide/cGMP pathway on CRT translocation. HT29 and HT29-dx cells were treated for 1 hr with fresh medium ( CTRL ), SNAP (100 μmol/l, SN ), 8-Br-cGMP (10 μmol/l, 8Br ), ODQ (10 μmol/l, ODQ ) and Rp-8-Br-cGMPS (10 μmol/l, Rp8Br ), in different combinations, then subjected to the following investigations. (A) The percentage of cells positive for surface CRT was measured in duplicate by FACS analysis as described in ‘Materials and methods’. Data are presented as means ± S.E. ( n = 3). Vs CTRL HT29: * P < 0.001; versus SN: ° P < 0.002. (B) Biotinylated surface CRT and total CRT were measured in Western blotting experiments, as reported in ‘Materials and methods’. The figure is representative of three experiments with similar results. The band density ratio between surface and total CRT was expressed as arbitrary units. Vs CTRL HT29: * P < 0.001; versus SN: ° P < 0.02. (C) Western blot analysis of VASP and phospho(Ser239)-VASP was performed on cytosolic extracts as described under ‘Materials and methods’. The figure is representative of three experiments with similar results. The band density ratio between phospho(Ser239)-VASP and total VASP was expressed as arbitrary units. Vs CTRL HT29: * P < 0.01; versus SN: ° P < 0.02.

Article Snippet: A total of 25 μg cell proteins were separated by SDS-PAGE, transferred to PVDF membrane sheets and probed with an anti-vasodilator-stimulated phosphoprotein (VASP) (rabbit polyclonal, diluted 1:500 in PBS-BSA 1%; Cell Signaling Technology, Inc., Danvers, MA, USA) or an anti-phospho(Ser 239)-VASP (mouse monoclonal, diluted 1:500 in PBS non-fat dry milk 3%, Millipore) antibody.

Techniques: Translocation Assay, Western Blot