Journal: Journal of Virology

Article Title: Human Immunodeficiency Virus-Associated Exosomes Promote Kaposi’s Sarcoma-Associated Herpesvirus Infection via the Epidermal Growth Factor Receptor

doi: 10.1128/jvi.01782-19

Figure Lengend Snippet: Fig. 2. HIV+ exosomes promote KSHV infection in oral epithelial cells. (A) Serial dilution of KSHV 845

Article Snippet: ; CD63 (clone NKI/C3, Cat# 468 NBP2-32819) and KSHV K8 (clone 8C12G10G1, NB-100-1086) antibodies were from Novus 469 Biol.

Techniques: Infection, Serial Dilution

Journal: Journal of Virology

Article Title: Human Immunodeficiency Virus-Associated Exosomes Promote Kaposi’s Sarcoma-Associated Herpesvirus Infection via the Epidermal Growth Factor Receptor

doi: 10.1128/jvi.01782-19

Figure Lengend Snippet: Fig. 4. HIV+ exosomes increase KSHV infection in oral mucosal tissue cultures. (A) Hematoxylin 874

Article Snippet: ; CD63 (clone NKI/C3, Cat# 468 NBP2-32819) and KSHV K8 (clone 8C12G10G1, NB-100-1086) antibodies were from Novus 469 Biol.

Techniques: Infection

Journal: Journal of Virology

Article Title: Human Immunodeficiency Virus-Associated Exosomes Promote Kaposi’s Sarcoma-Associated Herpesvirus Infection via the Epidermal Growth Factor Receptor

doi: 10.1128/jvi.01782-19

Figure Lengend Snippet: Fig. 5. HIV TAR is critical for HIV+ exosome-enhanced KSHV infection. (A) Immunoblots of HIV 889

Article Snippet: ; CD63 (clone NKI/C3, Cat# 468 NBP2-32819) and KSHV K8 (clone 8C12G10G1, NB-100-1086) antibodies were from Novus 469 Biol.

Techniques: Infection, Western Blot

Journal: Nature Communications

Article Title: Ubiquitination switches EphA2 vesicular traffic from a continuous safeguard to a finite signalling mode

doi: 10.1038/ncomms9047

Figure Lengend Snippet: ( a ) Colocalization of EphA2 and Rab11a. Left column: immunostaining of endogenous EphA2 (upper) and Rab11a (middle) in fixed Cos-7 cells, green/magenta overlay (lower). Right column: fluorescence images of ectopically expressed EphA2-mCitrine (first) and bfp–Rab11a (second) in living Cos-7 cells, green/magenta overlay (third). Fourth row represents magnified insets from respective overlay ROIs (yellow boxes). ( b ) Fluorescence redistribution after photoactivation of EphA2–paGFP in Cos-7 cells. First row: EphA2–paGFP fluorescence at the indicated times (s) after photoactivation on the RE, second: EphA2-mCherry fluorescence, third: bfp–Rab11a fluorescence. Lower graphs: ratiometrically determined decrease of EphA2–paGFP/EphA2-mCherry fluorescence at the RE (left) and gain at the PM (right). An average of 14 independent fluorescence loss and recovery curves (grey) were fitted to an exponential function to retrieve half-times ( t 1/2 ). Scale bars: 10 μm.

Article Snippet: Both Rab11a siRNA: sc-36340-SH (Santa Cruz Biotechnology) or scrambled non-targeting control siRNA: sc-37007 (Santa Cruz Biotechnology) was used at a final concentration of 50 nM for 72 h before validation of knockdown.

Techniques: Immunostaining, Fluorescence

Journal: Nature Communications

Article Title: Ubiquitination switches EphA2 vesicular traffic from a continuous safeguard to a finite signalling mode

doi: 10.1038/ncomms9047

Figure Lengend Snippet: ( a ) Ectopic expression of Rab11a or PTP1B suppresses autonomous activation of LIFEA2 in Cos-7 cells. For each panel: LIFEA2 fluorescence (first column), bfp–Rab11a or bfp–PTP1B fluorescence (second), overlay of LIFEA2 (green) and bfp–Rab11a or bfp–PTP1B (magenta) (third) and LIFEA2 τ -maps (fourth), color-coding, right. Right graphs: τ -histograms from n =3 experiments without ( n =7 cells) or with bfp–Rab11a (upper, n =8 cells) or bfp–PTP1B (lower, n =7 cells) expression. ( b ) Expression level dependence of LIFEA2 fluorescence lifetime ( τ ) on ectopic expression of bfp–Rab11a. 2D histogram of τ versus integrated fluorescence intensity ( F ) for individual cells ( n =54 cells) pre- (dark grey circles) and 20 min post-stimulation (light grey circles). ( c ) EphA2 phosphorylation is increased by Rab11a knockdown Left: western blot of Cos-7 lysates 72 h post-transfection with non-targeting siRNA (Ctrl, first column) or Rab11a siRNA (siRab11a, second column). Blots were probed for phospho-tyrosines (PY72, first row), EphA2 (second row), Rab11a (third row) and GAPDH (fourth row). Graph: ratio of Rab11a to GAPDH ( n =5), and PY72 to total EphA2 ( n =4) [mean±s.e.m., ** P <0.01; **** P <0.0001; unpaired t -test]. ( d ) Expression level dependence of LIFEA2 fluorescence lifetime ( τ ) on Rab11a knockdown in Cos-7 cells. 2D histogram of LIFEA2 τ versus its integrated fluorescence intensity ( F ) for individual cells ( n =49 cells) with non-targeting siRNA (Ctrl) or Rab11a siRNA (siRab11a). The two distributions fitted with an exponential function (black and blue lines) showed a statistical difference with P value=0.034 [* P <0.05; Kolmogorov–Smirnov test]. ( e ) Interaction of EphA2-mCitrine with substrate-trapping mutant PTP1B D/A-mCherry detected by FLIM–FRET in the absence (upper panel) or presence (lower panel) of bfp–Rab11a. First column: fluorescence images of EphA2-mCitrine, second: fluorescence images of mCherry–PTP1B D/A, third: τ (ns; color-coding right) of EphA2-mCitrine, fourth: fluorescence image of bfp–Rab11a. Graph: average fluorescence lifetime of EphA2-mCitrine ( τ )±s.e.m. versus its fluorescence intensity (FL) in absence (black, n =55 cells) or presence (red, n =50 cells) of bfp–Rab11a expression. The percentage of EphA2-mCitrine/PTP1B-D/A-mCherry interactions in the vicinity of Rab11a-positive endosomes (iii) was retrieved from the overlap between non-cell contact areas with low τ values (i, green) and high intensity areas in bfp–Rab11a images (ii, blue). Scale bars, 10 μm.

Article Snippet: Both Rab11a siRNA: sc-36340-SH (Santa Cruz Biotechnology) or scrambled non-targeting control siRNA: sc-37007 (Santa Cruz Biotechnology) was used at a final concentration of 50 nM for 72 h before validation of knockdown.

Techniques: Expressing, Activation Assay, Fluorescence, Phospho-proteomics, Knockdown, Western Blot, Transfection, Mutagenesis

Journal: Nature Communications

Article Title: Ubiquitination switches EphA2 vesicular traffic from a continuous safeguard to a finite signalling mode

doi: 10.1038/ncomms9047

Figure Lengend Snippet: ( a ) Time-lapse of EphA2 and Rab11a/Rab7 colocalization in Cos-7 cells on ephrinA1 stimulation. Left panels: fluorescence images of EphA2-mCitrine (left column), bfp–Rab11a (middle), mCherry-Rab7 (right), at indicated time in min. Right graphs: ratiometric analysis of EphA2-mCitrine (WT) and Y813F-EphA2-mCitrine (Y813F) localization on different membrane compartments, with and without ectopic expression of c-Cbl (Cbl). Upper: PM (PM-EphA2), middle: Rab11a-positive RE (RE-EphA2, inset: blow-up of the first 10 min), lower: Rab7-positive late endosome (LE-EphA2). Bar graphs: average amplitude between 48–53 min±s.e.m. ( n =12–18 cells from at least three independent experiments). Upper right box: color-coding. ( b ) Immunostaining of endogenous EphA2, Rab7 and LAMP1 in Cos-7 cells before (left column) and 20 min post-stimulation of Cos-7 cells with pre-clustered ephrinA1-Fc, 2 μg ml −1 (right column). EphA2 (first row), Rab7 (second row), EphA2 (green) and Rab7 (magenta) overlay (third row), LAMP1 (fourth row), EphA2 (green) and LAMP1 (magenta) overlay (fifth row). Scale bars: 10 μm. ( c ) c-Cbl induces EphA2 ubiquitination and degradation. Left: western blot of Cos-7 total cell lysate (Total, upper) and anti-GFP IP EphA2-mCitrine or Y813F-EphA2-mCitrine (IP, lower) showing time course of stimulation with pre-clustered ephrinA1-Fc (2 μg ml −1 ). Pre-clustered Fc fragment (Fc, 2 μg ml −1 ) was used as control (dashed box). c-Cbl-bfp and HA-Ubiquitin were co-expressed. Total lysates were probed with anti-EphA2, anti-c-Cbl and anti-GAPDH antibodies. IP was probed with anti-HA for ubiquitin (Ub) and anti-EphA2 antibodies. Right graphs: upper, ratiometric quantification of EphA2/GAPDH from blots of EphA2 (red) or Y813F-EphA2 (green) (mean±s.e.m. normalized to zero time point, n =3 blots). Lower: ratiometric quantification of Ub (ubiquitin)/EphA2 from 3 blots of anti-GFP IP EphA2 (red) or Y813F-EphA2 (green), right boxes: control Fc fragment (120 min).

Article Snippet: Both Rab11a siRNA: sc-36340-SH (Santa Cruz Biotechnology) or scrambled non-targeting control siRNA: sc-37007 (Santa Cruz Biotechnology) was used at a final concentration of 50 nM for 72 h before validation of knockdown.

Techniques: Fluorescence, Membrane, Expressing, Immunostaining, Ubiquitin Proteomics, Western Blot, Control