Review

rabbit anti rabgef1 (Novus Biologicals)

Novus Biologicals is a verified supplier

Novus Biologicals manufactures this product

About

News

Press Release

Team

Advisors

Partners

Contact

Bioz Stars

Bioz vStars

Buy from Supplier

Structured Review

Novus Biologicals rabbit anti rabgef1
$( A ) HeLa cells transiently expressing mChery-Parkin and GFP-mRABGEF1 were treated with DMSO or valinomycin for 3 hr followed by immunostaining. The magnified pictures were shown in the right. Bars, 10 μm. ( B ) Total cell lysates of ( A ) were analyzed by immunoblotting. Anti-GFP antibody was used for the GFP-mRABGEF1 detection. * and # denote ubiquitinated forms and truncated forms, respectively. ( C ) Quantification of <t>RABGEF1</t> recruitment to damaged mitochondria in ( A ). None, partial and complete denote that GFP-mRABGEF1 signals were overlapped with no, some of, and all mitochondria, respectively. ( D ) Recombinant ubiquitin (Ub) pre-treated with or without GST-TcPINK1 was subjected to pull-down assay with GST-mRABGEF1. W and E indicate wash and eluted fractions, respectively. 10%, 10% of input. ( E ) Percentages of the amount of ubiquitin in the eluted fraction in ( D ) were shown. The error bars represent mean ±SE from three independent experiments. ( F ) K48-linked and K63-linked Ub chains pre-treated with or without GST-TcPINK1 were subjected to pull-down assay with GST-mRABGEF1. ( G ) Interactions between GST-mRABGEF1 (WT or Y26A/A58D) and ubiquitin or phosphorylated ubiquitin were measured by ITC. N, stoichiometry of binding. 10.7554/eLife.31326.028 Figure 8—source data 1. Quantification of RABGEF1 recruitment to damaged mitochondria during mitophagy. 10.7554/eLife.31326.029 Figure 8—source data 2. Binding affinities of recombinant GST-mRABGEF1 with ubiquitin or phosphorylated ubiquitin. 10.7554/eLife.31326.030 Figure 8—source data 3. Binding affinities of recombinant GST-mRABGEF1 with ubiquitin or phosphorylated ubiquitin.$
Rabbit Anti Rabgef1, supplied by Novus Biologicals, used in various techniques. Bioz Stars score: 90/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/result/rabbit anti rabgef1/product/Novus Biologicals
Average 90 stars, based on 2 article reviews

rabbit anti rabgef1 - by Bioz Stars, 2026-06

90/100 stars

Images

1) Product Images from "Endosomal Rab cycles regulate Parkin-mediated mitophagy"

Article Title: Endosomal Rab cycles regulate Parkin-mediated mitophagy

Journal: eLife

doi: 10.7554/eLife.31326

$( A ) HeLa cells transiently expressing mChery-Parkin and GFP-mRABGEF1 were treated with DMSO or valinomycin for 3 hr followed by immunostaining. The magnified pictures were shown in the right. Bars, 10 μm. ( B ) Total cell lysates of ( A ) were analyzed by immunoblotting. Anti-GFP antibody was used for the GFP-mRABGEF1 detection. * and # denote ubiquitinated forms and truncated forms, respectively. ( C ) Quantification of RABGEF1 recruitment to damaged mitochondria in ( A ). None, partial and complete denote that GFP-mRABGEF1 signals were overlapped with no, some of, and all mitochondria, respectively. ( D ) Recombinant ubiquitin (Ub) pre-treated with or without GST-TcPINK1 was subjected to pull-down assay with GST-mRABGEF1. W and E indicate wash and eluted fractions, respectively. 10%, 10% of input. ( E ) Percentages of the amount of ubiquitin in the eluted fraction in ( D ) were shown. The error bars represent mean ±SE from three independent experiments. ( F ) K48-linked and K63-linked Ub chains pre-treated with or without GST-TcPINK1 were subjected to pull-down assay with GST-mRABGEF1. ( G ) Interactions between GST-mRABGEF1 (WT or Y26A/A58D) and ubiquitin or phosphorylated ubiquitin were measured by ITC. N, stoichiometry of binding. 10.7554/eLife.31326.028 Figure 8—source data 1. Quantification of RABGEF1 recruitment to damaged mitochondria during mitophagy. 10.7554/eLife.31326.029 Figure 8—source data 2. Binding affinities of recombinant GST-mRABGEF1 with ubiquitin or phosphorylated ubiquitin. 10.7554/eLife.31326.030 Figure 8—source data 3. Binding affinities of recombinant GST-mRABGEF1 with ubiquitin or phosphorylated ubiquitin.$
Figure Legend Snippet: ( A ) HeLa cells transiently expressing mChery-Parkin and GFP-mRABGEF1 were treated with DMSO or valinomycin for 3 hr followed by immunostaining. The magnified pictures were shown in the right. Bars, 10 μm. ( B ) Total cell lysates of ( A ) were analyzed by immunoblotting. Anti-GFP antibody was used for the GFP-mRABGEF1 detection. * and # denote ubiquitinated forms and truncated forms, respectively. ( C ) Quantification of RABGEF1 recruitment to damaged mitochondria in ( A ). None, partial and complete denote that GFP-mRABGEF1 signals were overlapped with no, some of, and all mitochondria, respectively. ( D ) Recombinant ubiquitin (Ub) pre-treated with or without GST-TcPINK1 was subjected to pull-down assay with GST-mRABGEF1. W and E indicate wash and eluted fractions, respectively. 10%, 10% of input. ( E ) Percentages of the amount of ubiquitin in the eluted fraction in ( D ) were shown. The error bars represent mean ±SE from three independent experiments. ( F ) K48-linked and K63-linked Ub chains pre-treated with or without GST-TcPINK1 were subjected to pull-down assay with GST-mRABGEF1. ( G ) Interactions between GST-mRABGEF1 (WT or Y26A/A58D) and ubiquitin or phosphorylated ubiquitin were measured by ITC. N, stoichiometry of binding. 10.7554/eLife.31326.028 Figure 8—source data 1. Quantification of RABGEF1 recruitment to damaged mitochondria during mitophagy. 10.7554/eLife.31326.029 Figure 8—source data 2. Binding affinities of recombinant GST-mRABGEF1 with ubiquitin or phosphorylated ubiquitin. 10.7554/eLife.31326.030 Figure 8—source data 3. Binding affinities of recombinant GST-mRABGEF1 with ubiquitin or phosphorylated ubiquitin.

Techniques Used: Expressing, Immunostaining, Western Blot, Recombinant, Ubiquitin Proteomics, Pull Down Assay, Binding Assay

( A ) The indicated cells were treated with DMSO or valinomycin for 3 hr followed by immunostaining. Bars, 10 μm. Graphs for quantification of RABGEF1 recruitment to mitochondria were shown below the images. None, partial and complete denote that GFP-mRABGEF1 signals were overlapped with no, some of, and all mitochondria, respectively. The error bars represent mean ±SE and over 100 cells were counted in each of three separate wells. ( B ) WT and TBC1D15/17 DKO HCT116 cells stably expressing mCherry-Parkin and GFP-mRABGEF1 were treated with DMSO or valinomycin for 3 hr. GFP-mRABGEF1 signals were enhanced by immunostaining with anti-GFP antibody. Bars, 10 μm. ( C ) Total cell lysates in ( B ) were analyzed by immunoblotting. * and # denote ubiquitinated forms and truncated forms, respectively. 10.7554/eLife.31326.031 Figure 8—figure supplement 1—source data 1. This excel file contains quantification of RABGEF1 (WT and Y26A/A58D mutant) recruitment to mitochondria in HCT116 (WT and TBC1D15/17 DKO) cells.

Figure Legend Snippet: ( A ) The indicated cells were treated with DMSO or valinomycin for 3 hr followed by immunostaining. Bars, 10 μm. Graphs for quantification of RABGEF1 recruitment to mitochondria were shown below the images. None, partial and complete denote that GFP-mRABGEF1 signals were overlapped with no, some of, and all mitochondria, respectively. The error bars represent mean ±SE and over 100 cells were counted in each of three separate wells. ( B ) WT and TBC1D15/17 DKO HCT116 cells stably expressing mCherry-Parkin and GFP-mRABGEF1 were treated with DMSO or valinomycin for 3 hr. GFP-mRABGEF1 signals were enhanced by immunostaining with anti-GFP antibody. Bars, 10 μm. ( C ) Total cell lysates in ( B ) were analyzed by immunoblotting. * and # denote ubiquitinated forms and truncated forms, respectively. 10.7554/eLife.31326.031 Figure 8—figure supplement 1—source data 1. This excel file contains quantification of RABGEF1 (WT and Y26A/A58D mutant) recruitment to mitochondria in HCT116 (WT and TBC1D15/17 DKO) cells.

Techniques Used: Immunostaining, Stable Transfection, Expressing, Western Blot, Mutagenesis

( A ) GFP-mRABGEF1 was transiently expressed in siRNA-treated HeLa cells. The cells were then treated with valinomycin for 3 hr followed by immunostaining. Bars, 20 μm. ( B ) Quantification of mitochondrial recruitment of GFP-mRABGEF1 in HeLa cells. ( C ) Quantification of mitochondrial recruitment of GFP-mRABGEF1 in HCT116 cells. None, partial and complete denote that GFP-mRABGEF1 signals were overlapped with no, some of, and all mitochondria, respectively. The error bars represent mean ± SE and over 100 cells were counted in each of three separate wells. 10.7554/eLife.31326.032 Figure 8—figure supplement 2—source data 2. Quantification of RABGEF1 recruitment to mitochondria in HeLa cells treated with the indicated siRNA during mitophagy. 10.7554/eLife.31326.033 Figure 8—figure supplement 2—source data 3. Quantification of RABGEF1 recruitment to mitochondria in HCT116 cells treated with the indicated siRNA during mitophagy.

Figure Legend Snippet: ( A ) GFP-mRABGEF1 was transiently expressed in siRNA-treated HeLa cells. The cells were then treated with valinomycin for 3 hr followed by immunostaining. Bars, 20 μm. ( B ) Quantification of mitochondrial recruitment of GFP-mRABGEF1 in HeLa cells. ( C ) Quantification of mitochondrial recruitment of GFP-mRABGEF1 in HCT116 cells. None, partial and complete denote that GFP-mRABGEF1 signals were overlapped with no, some of, and all mitochondria, respectively. The error bars represent mean ± SE and over 100 cells were counted in each of three separate wells. 10.7554/eLife.31326.032 Figure 8—figure supplement 2—source data 2. Quantification of RABGEF1 recruitment to mitochondria in HeLa cells treated with the indicated siRNA during mitophagy. 10.7554/eLife.31326.033 Figure 8—figure supplement 2—source data 3. Quantification of RABGEF1 recruitment to mitochondria in HCT116 cells treated with the indicated siRNA during mitophagy.

Techniques Used: Immunostaining

( A ) WT and RABGEF1-mAID HCT116 cells were treated with or without IAA for 16 hr. Total cell lysates were analyzed by immunoblotting. ( B ) Quantification of Parkin recruitment to mitochondria in WT and RABGEF1-mAID HCT116 cells after 3 hr of valinomycin treatment. Partial and complete denote that YFP-Parkin signals were overlapped with some of and all mitochondria, respectively. ( C ) YFP-Parkin stably expressing WT and RABGEF1-mAID HCT116 cells pre-treated with IAA were treated with valinomycin for the indicated times. Total cell lysates were analyzed by immunoblotting. ( D ) WT and RABGEF1-mAID HCT116 cells stably expressing YFP-Parkin and mt-mKeima were treated with IAA for 16 hr followed by DMSO or OAQ for 6 hr and subjected to FACS analysis. Plots are representative of n = 3 experiments. ( E ) Quantification of mitophagy in ( D ). Error bars represent mean ±SE of three independent experiments. Statistical differences were determined by student’s t-test. *p<0.05. 10.7554/eLife.31326.035 Figure 9—source data 1. Quantification of YFP-Parkin recruitment to mitochondria in RABGEF1-mAID HCT116 and the corresponding WT cells during mitophagy. 10.7554/eLife.31326.036 Figure 9—source data 2. Quantification of mitophagy using mt-mKeima and FACS analysis.

Figure Legend Snippet: ( A ) WT and RABGEF1-mAID HCT116 cells were treated with or without IAA for 16 hr. Total cell lysates were analyzed by immunoblotting. ( B ) Quantification of Parkin recruitment to mitochondria in WT and RABGEF1-mAID HCT116 cells after 3 hr of valinomycin treatment. Partial and complete denote that YFP-Parkin signals were overlapped with some of and all mitochondria, respectively. ( C ) YFP-Parkin stably expressing WT and RABGEF1-mAID HCT116 cells pre-treated with IAA were treated with valinomycin for the indicated times. Total cell lysates were analyzed by immunoblotting. ( D ) WT and RABGEF1-mAID HCT116 cells stably expressing YFP-Parkin and mt-mKeima were treated with IAA for 16 hr followed by DMSO or OAQ for 6 hr and subjected to FACS analysis. Plots are representative of n = 3 experiments. ( E ) Quantification of mitophagy in ( D ). Error bars represent mean ±SE of three independent experiments. Statistical differences were determined by student’s t-test. *p<0.05. 10.7554/eLife.31326.035 Figure 9—source data 1. Quantification of YFP-Parkin recruitment to mitochondria in RABGEF1-mAID HCT116 and the corresponding WT cells during mitophagy. 10.7554/eLife.31326.036 Figure 9—source data 2. Quantification of mitophagy using mt-mKeima and FACS analysis.

Techniques Used: Western Blot, Stable Transfection, Expressing

Figure Legend Snippet:

Techniques Used: Sequencing, Ubiquitin Proteomics, Protease Inhibitor, Western Blot, Recombinant, Software, Microscopy

Image Search Results

(A) The domain structure of RIN3. Red = proline-rich motifs. (B) Immunoblot analysis of RIN3 expression in human cell lines. 1:NIH3T3 w/RIN3 (+ctr), 2:NIH3T3 (−ctr), 3:HMC1 (mast), 4:LAD2 (mast), 5:THP1 (macrophage), 6:SaOs2 (osteoclast), 7:RAMOS (B cell), 8:K562 (myoblast), 9:Jurkat (T cell), 10: MCF10A (epithelial), 11:IMR90 (fibroblast), 12:U118 (glioblastoma). RIN3 appears as a double or single band depending on exposure time of the immunoblot. TUBB = β-tubulin. (C) Human mastocytosis sections stained using immunohistochemistry for RIN3 or secondary only as a control. Sections stained with anti-mast cell tryptase are shown to identify the infiltrating mast cells. (D) Lysates from three human mast cell lines (LAD2, LUVA, HMC1.1), an epithelial cell line (MCF10A), and a neuronal cell line (U118) were run on an SDS-PAGE gel and immunoblotted for RIN family members (RIN1/2/3) as well as RABGEF1, another GEF for RAB5.

Journal: PLoS ONE

Article Title: RIN3 Is a Negative Regulator of Mast Cell Responses to SCF

doi: 10.1371/journal.pone.0049615

Figure Lengend Snippet: (A) The domain structure of RIN3. Red = proline-rich motifs. (B) Immunoblot analysis of RIN3 expression in human cell lines. 1:NIH3T3 w/RIN3 (+ctr), 2:NIH3T3 (−ctr), 3:HMC1 (mast), 4:LAD2 (mast), 5:THP1 (macrophage), 6:SaOs2 (osteoclast), 7:RAMOS (B cell), 8:K562 (myoblast), 9:Jurkat (T cell), 10: MCF10A (epithelial), 11:IMR90 (fibroblast), 12:U118 (glioblastoma). RIN3 appears as a double or single band depending on exposure time of the immunoblot. TUBB = β-tubulin. (C) Human mastocytosis sections stained using immunohistochemistry for RIN3 or secondary only as a control. Sections stained with anti-mast cell tryptase are shown to identify the infiltrating mast cells. (D) Lysates from three human mast cell lines (LAD2, LUVA, HMC1.1), an epithelial cell line (MCF10A), and a neuronal cell line (U118) were run on an SDS-PAGE gel and immunoblotted for RIN family members (RIN1/2/3) as well as RABGEF1, another GEF for RAB5.

Article Snippet: Other antibodies, their sources and the dilutions used for immunoblot probing were: rabbit anti-RIN1 1/1,000; rabbit anti-RAB5 (Abcam ab18211) 1/250; rabbit anti-RAS (Novus Bio EP1125Y) 1/5000; rabbit anti-KIT (Cell Signaling 3392) 1/500; mouse anti-KIT (Abcam Ab81) 1/500; mouse anti-α tubulin (Sigma) 1/3000; mouse anti-KIT conjugated to PE/Cy7 (Biolegend 104D2) 1/100; rabbit anti-GAPDH (Abcam) 1/3000; mouse anti-pTyr clone 4G10 (Millipore) 1/500; rabbit anti-RABGEF1 (Sigma) 1/1,000; rabbit anti-BIN2 1/3000; mouse anti-transferrin receptor (Invitrogen) 1/500; anti MCT (DakyCytomation) 1/2000.

Techniques: Western Blot, Expressing, Staining, Immunohistochemistry, SDS Page

Alkalization of endosome acidity retained endocytic WSSV in the cytoplasm, resulting in reduced viral infection. (A) Endocytic WSSV virions were trapped in dysfunctional endosomes induced by alkalization. (a) WSSV and MVBs are indicated by white arrows and yellow arrows, respectively, under TEM observation. C, cytoplasm; N, nucleus. (b) The isolated WSSV virions (red) colocalized with endosomes (green), as identified by immunocytochemistry. The arrows indicate colocalization between WSSV virions and endosomes in the merged images. (c) The relative percentage of WSSV colocalized with the endosome was significantly higher in alkalized cells than in control cells, as shown by histogram analysis of the colocalization spots between VP28 and RabGEF1. CQ, chloroquine. (B) Acidic endosomes were accumulated by alkalization in Hpt cells. (a) Acidic endosomes, indicated by arrows, were dual labeled with WGA and LysoTracker staining. (b and c) Decreased fluorescence (LysoTracker staining) (b) and increased relative size of acidic endosomes (colocalization between WGA and LysoTracker staining) (c) were induced by alkalization with chloroquine or NH4Cl, respectively, for 1 h in Hpt cells, followed by examination by confocal microscopy. The results were statistically analyzed and presented by histogram analysis. (C) Endocytic WSSV virions were detained in the alkalized endosomes, as determined by real-time observation in live cells. (a) Fusion and accumulation of endocytic WSSV virions with endosomes. In control cells, DiD-WSSV virions (red) were fused with acidic endosomes labeled with LysoTracker (green), and endosomes containing DiD-WSSV gathered and accumulated, as indicated by the yellow lines, over time. (b) Most of the endocytic WSSV virions were isolated and retained in the alkalized endosomes. Hpt cells were pretreated with chloroquine, followed by infection with WSSV labeled with DiD (red). The acidic endosomes were labeled with LysoTracker (green). The arrows indicate accumulated WSSV virions without fusion in the significantly enlarged endosomes caused by alkalization. (c) Endocytic WSSV virions were accumulated and retained in enlarged endosomal vesicles induced by alkalization. DiD-WSSV and DiO-WSSV accumulated in alkalized endosomes, in which fusion between WSSV and endosomes was clearly reduced. The Hpt cells were pretreated with chloroquine, followed by simultaneous infection with WSSV labeled with DiO (green) and WSSV labeled with DiD (red). Accumulated WSSV virions in endosomal vesicles are indicated by dashed circles over time. (D) WSSV fusion was strongly inhibited by alkalizing acidic endosomes. (Left) Extensive colocalized fluorescence of the WSSV envelope and nucleocapsid, with yellow signal (indicated by arrows), was present in Hpt cells treated with the alkalizer chloroquine or NH4Cl. (Right) The relative percentage of viral envelope colocalized with the nucleocapsid was significantly higher in alkalized cells than in control cells, as shown by histogram analysis of the colocalization spots between VP28 and VP664. (E) Both degradation and replication of WSSV were reduced by alkalizing acidic endosomes. (a) Degradation of WSSV components was reduced by alkalizing acidic endosomes in Hpt cells. The degradation of WSSV components, as indicated by intracellular presence of viral envelope VP28 protein, was determined by immunoblotting against VP28 4 hpi in Hpt cells. (b) WSSV replication was significantly inhibited by alkalizing acidic endosomes in Hpt cells. (c) WSSV copy numbers were significantly reduced by alkalizing acidic endosomes in Hpt cells. (F) Both fusion and replication of WSSV were not inhibited by blocking endosomal maturation. (a) Endosome maturation did not affect WSSV fusion. No significant inhibition of WSSV fusion was found in YM-201636-treated cells, in contrast to positive-control cells exposed to bafilomycin-A1 (Baf-A1). Hpt cells were pretreated with YM-201636 to block endosome maturation or with bafilomycin-A1 to inhibit V-ATPase activity mediated by acidity within endosomes, followed by WSSV infection. WSSV fusion was determined by immunofluorescence assay against the colocalization between viral envelope protein VP28 and nucleocapsid protein VP664. (b) Blocking endosome maturation did not inhibit WSSV replication. Blocking endosome maturation with YM-201636 did not result in clear inhibition of WSSV replication in Hpt cells (top). In contrast, WSSV replication was significantly inhibited by bafilomycin-A1 exposure (bottom). TEM, confocal microscopy analysis, and real-time imaging were performed from 3 hpi unless otherwise stated. WSSV replication and copy numbers were evaluated by relative gene expression of VP28 transcript using qRT-PCR and by examination of VP28 DNA using PCR 24 hpi. n.s, no significant difference; *, P < 0.05; **, P < 0.01; ***, P < 0.001.

Journal: Journal of Virology

Article Title: White Spot Syndrome Virus Benefits from Endosomal Trafficking, Substantially Facilitated by a Valosin-Containing Protein, To Escape Autophagic Elimination and Propagate in the Crustacean Cherax quadricarinatus

doi: 10.1128/JVI.01570-20

Figure Lengend Snippet: Alkalization of endosome acidity retained endocytic WSSV in the cytoplasm, resulting in reduced viral infection. (A) Endocytic WSSV virions were trapped in dysfunctional endosomes induced by alkalization. (a) WSSV and MVBs are indicated by white arrows and yellow arrows, respectively, under TEM observation. C, cytoplasm; N, nucleus. (b) The isolated WSSV virions (red) colocalized with endosomes (green), as identified by immunocytochemistry. The arrows indicate colocalization between WSSV virions and endosomes in the merged images. (c) The relative percentage of WSSV colocalized with the endosome was significantly higher in alkalized cells than in control cells, as shown by histogram analysis of the colocalization spots between VP28 and RabGEF1. CQ, chloroquine. (B) Acidic endosomes were accumulated by alkalization in Hpt cells. (a) Acidic endosomes, indicated by arrows, were dual labeled with WGA and LysoTracker staining. (b and c) Decreased fluorescence (LysoTracker staining) (b) and increased relative size of acidic endosomes (colocalization between WGA and LysoTracker staining) (c) were induced by alkalization with chloroquine or NH4Cl, respectively, for 1 h in Hpt cells, followed by examination by confocal microscopy. The results were statistically analyzed and presented by histogram analysis. (C) Endocytic WSSV virions were detained in the alkalized endosomes, as determined by real-time observation in live cells. (a) Fusion and accumulation of endocytic WSSV virions with endosomes. In control cells, DiD-WSSV virions (red) were fused with acidic endosomes labeled with LysoTracker (green), and endosomes containing DiD-WSSV gathered and accumulated, as indicated by the yellow lines, over time. (b) Most of the endocytic WSSV virions were isolated and retained in the alkalized endosomes. Hpt cells were pretreated with chloroquine, followed by infection with WSSV labeled with DiD (red). The acidic endosomes were labeled with LysoTracker (green). The arrows indicate accumulated WSSV virions without fusion in the significantly enlarged endosomes caused by alkalization. (c) Endocytic WSSV virions were accumulated and retained in enlarged endosomal vesicles induced by alkalization. DiD-WSSV and DiO-WSSV accumulated in alkalized endosomes, in which fusion between WSSV and endosomes was clearly reduced. The Hpt cells were pretreated with chloroquine, followed by simultaneous infection with WSSV labeled with DiO (green) and WSSV labeled with DiD (red). Accumulated WSSV virions in endosomal vesicles are indicated by dashed circles over time. (D) WSSV fusion was strongly inhibited by alkalizing acidic endosomes. (Left) Extensive colocalized fluorescence of the WSSV envelope and nucleocapsid, with yellow signal (indicated by arrows), was present in Hpt cells treated with the alkalizer chloroquine or NH4Cl. (Right) The relative percentage of viral envelope colocalized with the nucleocapsid was significantly higher in alkalized cells than in control cells, as shown by histogram analysis of the colocalization spots between VP28 and VP664. (E) Both degradation and replication of WSSV were reduced by alkalizing acidic endosomes. (a) Degradation of WSSV components was reduced by alkalizing acidic endosomes in Hpt cells. The degradation of WSSV components, as indicated by intracellular presence of viral envelope VP28 protein, was determined by immunoblotting against VP28 4 hpi in Hpt cells. (b) WSSV replication was significantly inhibited by alkalizing acidic endosomes in Hpt cells. (c) WSSV copy numbers were significantly reduced by alkalizing acidic endosomes in Hpt cells. (F) Both fusion and replication of WSSV were not inhibited by blocking endosomal maturation. (a) Endosome maturation did not affect WSSV fusion. No significant inhibition of WSSV fusion was found in YM-201636-treated cells, in contrast to positive-control cells exposed to bafilomycin-A1 (Baf-A1). Hpt cells were pretreated with YM-201636 to block endosome maturation or with bafilomycin-A1 to inhibit V-ATPase activity mediated by acidity within endosomes, followed by WSSV infection. WSSV fusion was determined by immunofluorescence assay against the colocalization between viral envelope protein VP28 and nucleocapsid protein VP664. (b) Blocking endosome maturation did not inhibit WSSV replication. Blocking endosome maturation with YM-201636 did not result in clear inhibition of WSSV replication in Hpt cells (top). In contrast, WSSV replication was significantly inhibited by bafilomycin-A1 exposure (bottom). TEM, confocal microscopy analysis, and real-time imaging were performed from 3 hpi unless otherwise stated. WSSV replication and copy numbers were evaluated by relative gene expression of VP28 transcript using qRT-PCR and by examination of VP28 DNA using PCR 24 hpi. n.s, no significant difference; *, P < 0.05; **, P < 0.01; ***, P < 0.001.

Article Snippet: Rabbit anti-RabGEF1 polyclonal antibody was purchased from ABclonal (United States).

Techniques: Infection, Isolation, Immunocytochemistry, Labeling, Staining, Fluorescence, Confocal Microscopy, Western Blot, Blocking Assay, Inhibition, Positive Control, Activity Assay, Immunofluorescence, Imaging, Expressing, Quantitative RT-PCR

WSSV replication via endosomal trafficking was disrupted by blocking CqVCP ATPase activity. (A) Both CqVCP gene and protein expression were responsive to WSSV infection in vivo. The transcript expression (a) and protein (b) levels of CqVCP were profoundly induced in crayfish Hpt tissue in vivo 1, 6, and 12 h after WSSV infection. (B) WSSV infection was significantly inhibited by disrupting CqVCP ATPase activity. (a) Both replication and degradation of WSSV were significantly decreased by CqVCP gene silencing. (Left) CqVCP gene silencing resulted in significant inhibition of CqVCP gene expression, which was accompanied by profoundly reduced viral replication. (Right) The CqVCP protein level was reduced by gene silencing of CqVCP, followed by substantially decreased VP28 degradation. (b) Both replication and degradation of WSSV were significantly inhibited by blocking CqVCP ATPase activity with DBeQ exposure. (C) Dynamic analysis of accumulated dysfunctional endosomes enclosing WSSV after disrupting CqVCP ATPase activity. The observation of intracellular trafficking of WSSV over time was done from 3 hpi in Hpt cells. (Top) The arrows indicate colocalization of DiD-WSSV within acidic endosomes moving toward the cell perinuclear area in control cells. (Bottom) DiD-WSSV was colocalized within dysfunctional endosomes caused by disrupting CqVCP ATPase activity with DBeQ exposure, which further accumulated to form enlarged vesicles, as indicated by the arrows. Acidic endosomes were dual labeled with WGA and LysoTracker staining. (D) WSSV fusion was severely blocked in dysfunctional endosomes caused by disrupting CqVCP ATPase activity. (a) CqVCP accumulated and colocalized with WSSV aggregates in dysfunctional endosomes. CqVCP was found to be colocalized with both RabGEF1 (top) and WSSV (bottom) by immunostaining against VP28, as indicated by the arrows, in Hpt cells after disrupting CqVCP ATPase activity by DBeQ exposure. In contrast, no significant accumulated colocalization of CqVCP and RabGEF1 or of CqVCP and WSSV was present in dimethyl sulfoxide (DMSO)-treated control Hpt cells. (b) WSSV fusion was significantly inhibited by blocking CqVCP ATPase activity. (Top) WSSV was colocalized with endosomes, as shown by immunostaining with anti-RabGEF1 antibody. (Bottom) Extensive colocalization of WSSV envelope protein VP28 and nucleocapsid protein (as indicated by immunostaining against VP664) was present in Hpt cells lacking CqVCP ATPase activity caused by DBeQ exposure. No significant colocalization of VP28 and RabGEF1 or of VP28 and VP664 was present in the DMSO-treated control cells due to the degradation of the detached viral envelope over time. Colocalization is indicated by the arrows. (Right) Fluorescence intensity colocalization of VP28 and RabGEF1 or of VP28 and VP664 was analyzed as shown by histogram analysis. (c) Aggregation of WSSV was strongly increased in dysfunctional endosomes caused by blocking CqVCP ATPase activity. The ratio of WSSV aggregates (≥3 or <3 virions per endosome) was relatively quantified with at least 80 cells containing WSSV in each group. Aggregated WSSV virions are indicated by white arrows, and endosomes containing WSSV virions are indicated by yellow arrows. The assays were performed 4 hpi. (E) Both degradation and replication of WSSV were strongly inhibited by trapping of virions within dysfunctional endosomes caused by blocking CqVCP ATPase activity. (a) Degradation of WSSV was strongly inhibited by blocking of CqVCP ATPase activity. (b) Replication of WSSV was strongly inhibited by blocking of CqVCP ATPase activity. Hpt cells were preexposed to cycloheximide to block protein translation activity needed for viral replication, followed by WSSV infection. (F) Propagation of progeny WSSV virions was abolished by blocking CqVCP ATPase activity. (Top) Progeny WSSV virions, (yellow arrows) were mostly present in the nuclear area in control Hpt cells 18 hpi, but fewer were found in the Hpt cells after blocking CqVCP ATPase activity by DBeQ exposure. Segregated endocytic WSSV virions or retained viral components, such as VP28 and VP664 (white arrows), were mainly present in the cytoplasm of Hpt cells exposed to DBeQ. (Bottom) Progeny virions (yellow arrows) were found in control cells, but rarely in the Hpt cells exposed to DBeQ, by TEM analysis 18 hpi. The retained viral components were found to be present in endocytic vesicles in the cytoplasm (white arrows) in DBeQ-exposed cells. WSSV replication was determined by quantification of the relative gene expression of VP28 by qRT-PCR. The degradation of WSSV was determined by immunoblotting against the presence of envelope protein VP28 with anti-VP28 monoclonal antibody. Intracellular localization of WSSV in Hpt cells was performed by TEM, where CqVCP ATPase activity was blocked by DBeQ exposure. N, nucleus; C, cytoplasm. n.s, no significant difference; *, P < 0.05; **, P < 0.01; ***, P < 0.001.

Journal: Journal of Virology

doi: 10.1128/JVI.01570-20

Figure Lengend Snippet: WSSV replication via endosomal trafficking was disrupted by blocking CqVCP ATPase activity. (A) Both CqVCP gene and protein expression were responsive to WSSV infection in vivo. The transcript expression (a) and protein (b) levels of CqVCP were profoundly induced in crayfish Hpt tissue in vivo 1, 6, and 12 h after WSSV infection. (B) WSSV infection was significantly inhibited by disrupting CqVCP ATPase activity. (a) Both replication and degradation of WSSV were significantly decreased by CqVCP gene silencing. (Left) CqVCP gene silencing resulted in significant inhibition of CqVCP gene expression, which was accompanied by profoundly reduced viral replication. (Right) The CqVCP protein level was reduced by gene silencing of CqVCP, followed by substantially decreased VP28 degradation. (b) Both replication and degradation of WSSV were significantly inhibited by blocking CqVCP ATPase activity with DBeQ exposure. (C) Dynamic analysis of accumulated dysfunctional endosomes enclosing WSSV after disrupting CqVCP ATPase activity. The observation of intracellular trafficking of WSSV over time was done from 3 hpi in Hpt cells. (Top) The arrows indicate colocalization of DiD-WSSV within acidic endosomes moving toward the cell perinuclear area in control cells. (Bottom) DiD-WSSV was colocalized within dysfunctional endosomes caused by disrupting CqVCP ATPase activity with DBeQ exposure, which further accumulated to form enlarged vesicles, as indicated by the arrows. Acidic endosomes were dual labeled with WGA and LysoTracker staining. (D) WSSV fusion was severely blocked in dysfunctional endosomes caused by disrupting CqVCP ATPase activity. (a) CqVCP accumulated and colocalized with WSSV aggregates in dysfunctional endosomes. CqVCP was found to be colocalized with both RabGEF1 (top) and WSSV (bottom) by immunostaining against VP28, as indicated by the arrows, in Hpt cells after disrupting CqVCP ATPase activity by DBeQ exposure. In contrast, no significant accumulated colocalization of CqVCP and RabGEF1 or of CqVCP and WSSV was present in dimethyl sulfoxide (DMSO)-treated control Hpt cells. (b) WSSV fusion was significantly inhibited by blocking CqVCP ATPase activity. (Top) WSSV was colocalized with endosomes, as shown by immunostaining with anti-RabGEF1 antibody. (Bottom) Extensive colocalization of WSSV envelope protein VP28 and nucleocapsid protein (as indicated by immunostaining against VP664) was present in Hpt cells lacking CqVCP ATPase activity caused by DBeQ exposure. No significant colocalization of VP28 and RabGEF1 or of VP28 and VP664 was present in the DMSO-treated control cells due to the degradation of the detached viral envelope over time. Colocalization is indicated by the arrows. (Right) Fluorescence intensity colocalization of VP28 and RabGEF1 or of VP28 and VP664 was analyzed as shown by histogram analysis. (c) Aggregation of WSSV was strongly increased in dysfunctional endosomes caused by blocking CqVCP ATPase activity. The ratio of WSSV aggregates (≥3 or <3 virions per endosome) was relatively quantified with at least 80 cells containing WSSV in each group. Aggregated WSSV virions are indicated by white arrows, and endosomes containing WSSV virions are indicated by yellow arrows. The assays were performed 4 hpi. (E) Both degradation and replication of WSSV were strongly inhibited by trapping of virions within dysfunctional endosomes caused by blocking CqVCP ATPase activity. (a) Degradation of WSSV was strongly inhibited by blocking of CqVCP ATPase activity. (b) Replication of WSSV was strongly inhibited by blocking of CqVCP ATPase activity. Hpt cells were preexposed to cycloheximide to block protein translation activity needed for viral replication, followed by WSSV infection. (F) Propagation of progeny WSSV virions was abolished by blocking CqVCP ATPase activity. (Top) Progeny WSSV virions, (yellow arrows) were mostly present in the nuclear area in control Hpt cells 18 hpi, but fewer were found in the Hpt cells after blocking CqVCP ATPase activity by DBeQ exposure. Segregated endocytic WSSV virions or retained viral components, such as VP28 and VP664 (white arrows), were mainly present in the cytoplasm of Hpt cells exposed to DBeQ. (Bottom) Progeny virions (yellow arrows) were found in control cells, but rarely in the Hpt cells exposed to DBeQ, by TEM analysis 18 hpi. The retained viral components were found to be present in endocytic vesicles in the cytoplasm (white arrows) in DBeQ-exposed cells. WSSV replication was determined by quantification of the relative gene expression of VP28 by qRT-PCR. The degradation of WSSV was determined by immunoblotting against the presence of envelope protein VP28 with anti-VP28 monoclonal antibody. Intracellular localization of WSSV in Hpt cells was performed by TEM, where CqVCP ATPase activity was blocked by DBeQ exposure. N, nucleus; C, cytoplasm. n.s, no significant difference; *, P < 0.05; **, P < 0.01; ***, P < 0.001.

Article Snippet: Rabbit anti-RabGEF1 polyclonal antibody was purchased from ABclonal (United States).

Techniques: Blocking Assay, Activity Assay, Expressing, Infection, In Vivo, Inhibition, Labeling, Staining, Immunostaining, Fluorescence, Quantitative RT-PCR, Western Blot

Increased autophagic activity substantially reduced WSSV infection. (A) Autophagy was increased by WSSV infection. (Left) Both protein expression of CqGABARAP and conversion of CqGABARAP-I to CqGABARAP-II were induced by WSSV infection. (Right) The relative quantitation of the conversion of CqGABARAP-I to CqGABARAP-II was determined by histogram analysis. (B) Autophagy was induced by AKTi 1/2 exposure in Hpt cells. (a) Formation of CqGABARAP puncta was increased against AKTi 1/2 exposure in a dose-dependent manner. (b) Statistical analysis of formation of fluorescent CqGABARAP puncta. The relative increase in fluorescence of CqGABARAP puncta in subpanel a was quantified as presented by histogram analysis. (c) Significant conversion of CqGABARAP-I to CqGABARAP-II was induced by AKTi 1/2 in a dose-dependent manner. (C) Real-time observation of increased accumulation and fusion of WSSV-containing endosomes caused by induced autophagic activity. (Top) Fusion of WSSV-containing endosomes (white arrows) in control cells. (Bottom) Accumulation of WSSV-containing endosomes (yellow arrows) was clearly increased by induced autophagic activity against AKTi 1/2 exposure in Hpt cells. Hpt cells were infected with WSSV labeled with DiD, and endosomes were labeled by WGA staining. Time, 3 hpi. (D) WSSV envelope fusion was significantly inhibited by increased autophagic activity. (a) Accumulation and fusion of WSSV-containing endosomes with autophagosomes were increased by induced autophagic activity. More endosomes containing WSSV virions accumulated and fused with each other to form enlarged vesicles (top, arrows), which were surrounded by autophagosomes (bottom, arrows) in Hpt cells with increased autophagic activity caused by AKTi 1/2 exposure. Colocalization of endosomes with WSSV (top) or autophagosomes and endosomes (bottom), as indicated by the arrows, was increased by induced autophagic activity. (b) Release of WSSV nucleocapsid was significantly inhibited by increased autophagic activity. Colocalization of autophagosomes with WSSV (top) or VP28 with VP664 (bottom), as indicated by the arrows, was significantly increased in Hpt cells with higher autophagic activity induced by AKTi 1/2 exposure (left), and the relative colocalization efficiency was determined by histogram analysis (right). (a and b) WSSV was localized with anti-VP28 antibody against viral envelope protein or with anti-VP664 antibody against nucleocapsid protein. Endosomes were immunostained by anti-RabGEF1 antibody or labeled with WGA fluorescent dye, respectively. Autophagosomes were localized with anti-CqGABARAP antibody. (c) Accumulation of endocytic WSSV virions in autophagosomes was increased by induced autophagic activity. (Left) Autophagosomes with double-layer membranes are indicated by yellow arrows, and the enclosed WSSV virions are indicated by white arrows. (Right) The numbers of WSSV-containing autophagosomes with <3 or >3 virions per autophagosome were calculated in AKTi 1/2-exposed cells and control cells, respectively, as shown by histogram analysis. Hpt cells were preexposed with AKTi 1/2, followed by WSSV infection. Cell samples were analyzed by TEM, and the numbers of WSSV-containing autophagosomes were defined as <3 and ≥3 virions per autophagosome. (E) Reduced WSSV replication due to increased viral degradation by induced autophagic activity. (a) Hpt cells were preexposed to cycloheximide (CHX) to block protein translation activity or to AKTi 1/2 to increase autophagic activity, as indicated, and subsequently infected with WSSV. The presence of WSSV components was determined by immunoblotting against viral envelope protein VP28. (b) WSSV replication was evaluated by relative gene expression of VP28 in Hpt cells using qRT-PCR. (F) Both acidity and acidic-enzyme activity were significantly increased in Hpt cells by induced autophagic activity. (Top) Accumulation of autophagosomes and acidity of intracellular acidic vesicles, including autophagosome, were strongly increased by autophagic activity induced by AKTi 1/2 exposure in Hpt cells. (Left) Acidic vesicles were stained with LysoTracker. The arrows indicate accumulated acidic vesicles, which are likely to increase the efficiency of autophagic degradation. (Right) Histograms showing substantially increased accumulation of autophagosomes induced by increased autophagy activity. (Bottom) Fluorescent signal of cathepsin-magic dye, indicating the acidity-dependent enzyme activity, such as cathepsin L activity, in intracellular acidic vesicles, was profoundly increased in Hpt cells with autophagic activity induced by AKTi 1/2 exposure. (Left) The arrows indicate accumulated acidic vesicles with higher cathepsin L enzyme activity, which likely induced the efficiency of autophagic degradation. (Right) Histograms showing significantly higher fluorescent intensity of acidic vesicles induced by increased autophagy activity. The confocal microscopy analysis with WSSV infection was performed from 4 hpi unless otherwise stated. n.s, no significant difference; *, P < 0.05; **, P < 0.01; ***, P < 0.001.

Journal: Journal of Virology

doi: 10.1128/JVI.01570-20

Figure Lengend Snippet: Increased autophagic activity substantially reduced WSSV infection. (A) Autophagy was increased by WSSV infection. (Left) Both protein expression of CqGABARAP and conversion of CqGABARAP-I to CqGABARAP-II were induced by WSSV infection. (Right) The relative quantitation of the conversion of CqGABARAP-I to CqGABARAP-II was determined by histogram analysis. (B) Autophagy was induced by AKTi 1/2 exposure in Hpt cells. (a) Formation of CqGABARAP puncta was increased against AKTi 1/2 exposure in a dose-dependent manner. (b) Statistical analysis of formation of fluorescent CqGABARAP puncta. The relative increase in fluorescence of CqGABARAP puncta in subpanel a was quantified as presented by histogram analysis. (c) Significant conversion of CqGABARAP-I to CqGABARAP-II was induced by AKTi 1/2 in a dose-dependent manner. (C) Real-time observation of increased accumulation and fusion of WSSV-containing endosomes caused by induced autophagic activity. (Top) Fusion of WSSV-containing endosomes (white arrows) in control cells. (Bottom) Accumulation of WSSV-containing endosomes (yellow arrows) was clearly increased by induced autophagic activity against AKTi 1/2 exposure in Hpt cells. Hpt cells were infected with WSSV labeled with DiD, and endosomes were labeled by WGA staining. Time, 3 hpi. (D) WSSV envelope fusion was significantly inhibited by increased autophagic activity. (a) Accumulation and fusion of WSSV-containing endosomes with autophagosomes were increased by induced autophagic activity. More endosomes containing WSSV virions accumulated and fused with each other to form enlarged vesicles (top, arrows), which were surrounded by autophagosomes (bottom, arrows) in Hpt cells with increased autophagic activity caused by AKTi 1/2 exposure. Colocalization of endosomes with WSSV (top) or autophagosomes and endosomes (bottom), as indicated by the arrows, was increased by induced autophagic activity. (b) Release of WSSV nucleocapsid was significantly inhibited by increased autophagic activity. Colocalization of autophagosomes with WSSV (top) or VP28 with VP664 (bottom), as indicated by the arrows, was significantly increased in Hpt cells with higher autophagic activity induced by AKTi 1/2 exposure (left), and the relative colocalization efficiency was determined by histogram analysis (right). (a and b) WSSV was localized with anti-VP28 antibody against viral envelope protein or with anti-VP664 antibody against nucleocapsid protein. Endosomes were immunostained by anti-RabGEF1 antibody or labeled with WGA fluorescent dye, respectively. Autophagosomes were localized with anti-CqGABARAP antibody. (c) Accumulation of endocytic WSSV virions in autophagosomes was increased by induced autophagic activity. (Left) Autophagosomes with double-layer membranes are indicated by yellow arrows, and the enclosed WSSV virions are indicated by white arrows. (Right) The numbers of WSSV-containing autophagosomes with <3 or >3 virions per autophagosome were calculated in AKTi 1/2-exposed cells and control cells, respectively, as shown by histogram analysis. Hpt cells were preexposed with AKTi 1/2, followed by WSSV infection. Cell samples were analyzed by TEM, and the numbers of WSSV-containing autophagosomes were defined as <3 and ≥3 virions per autophagosome. (E) Reduced WSSV replication due to increased viral degradation by induced autophagic activity. (a) Hpt cells were preexposed to cycloheximide (CHX) to block protein translation activity or to AKTi 1/2 to increase autophagic activity, as indicated, and subsequently infected with WSSV. The presence of WSSV components was determined by immunoblotting against viral envelope protein VP28. (b) WSSV replication was evaluated by relative gene expression of VP28 in Hpt cells using qRT-PCR. (F) Both acidity and acidic-enzyme activity were significantly increased in Hpt cells by induced autophagic activity. (Top) Accumulation of autophagosomes and acidity of intracellular acidic vesicles, including autophagosome, were strongly increased by autophagic activity induced by AKTi 1/2 exposure in Hpt cells. (Left) Acidic vesicles were stained with LysoTracker. The arrows indicate accumulated acidic vesicles, which are likely to increase the efficiency of autophagic degradation. (Right) Histograms showing substantially increased accumulation of autophagosomes induced by increased autophagy activity. (Bottom) Fluorescent signal of cathepsin-magic dye, indicating the acidity-dependent enzyme activity, such as cathepsin L activity, in intracellular acidic vesicles, was profoundly increased in Hpt cells with autophagic activity induced by AKTi 1/2 exposure. (Left) The arrows indicate accumulated acidic vesicles with higher cathepsin L enzyme activity, which likely induced the efficiency of autophagic degradation. (Right) Histograms showing significantly higher fluorescent intensity of acidic vesicles induced by increased autophagy activity. The confocal microscopy analysis with WSSV infection was performed from 4 hpi unless otherwise stated. n.s, no significant difference; *, P < 0.05; **, P < 0.01; ***, P < 0.001.

Article Snippet: Rabbit anti-RabGEF1 polyclonal antibody was purchased from ABclonal (United States).

Techniques: Activity Assay, Infection, Expressing, Quantitation Assay, Fluorescence, Labeling, Staining, Blocking Assay, Western Blot, Quantitative RT-PCR, Confocal Microscopy

WSSV escaped from autophagic elimination via direction to the endosomal delivery system, substantially facilitated by CqVCP. (A) Most endocytic WSSV virions escaped from autophagy degradation via direction to endosomes, facilitated by CqVCP. (a) Dysfunctional endosomes, caused by blocking CqVCP ATPase activity, resulted in more WSSV aggregation than was caused by increasing autophagic activity. Aggregated WSSV virions were localized by immunostaining against viral envelope protein VP28 (white arrows). CqVCP ATPase activity was blocked by DBeQ exposure, and increased autophagic activity was induced by AKTi 1/2 exposure in Hpt cells. (b) Relative quantification of the integrated optical density of fluorescent VP28 signal (a, top row). (c) Relative quantification of colocalization of VP28 and VP664. Histogram analysis shows the integrated optical density of the colocalized fluorescent signals of VP28 and VP664 in Hpt cells (a, bottom row, yellow arrows). (d) Dysfunctional endosomes, caused by blocking CqVCP ATPase activity, resulted in more inhibition of WSSV replication than was caused by increasing autophagic activity. The relative gene expression of WSSV was determined by the presence of VP28 transcript 24 hpi in Hpt cells, using qRT-PCR. (B) Homotypic fusion of WSSV-containing endocytic vesicles and endosomes was significantly inhibited by restricting D2 in CqVCP in an ADP-bound state. (a) WSSV envelope fusion was profoundly inhibited by blocking homotypic fusion of WSSV-containing endocytic vesicles and endosomes. (Top) Inhibition of viral fusion was indirectly indicated by fluorescence of WSSV envelope protein VP28 (arrows). (Left) In contrast to control Hpt cells after viral fusion, much more fluorescent viral envelope protein, such as VP28, was present in cells lacking CqVCP activity caused by NMS873 exposure. (Right) Histogram analysis showing fluorescence intensity of intracellular viral envelope VP28 protein. (Bottom) Homotypic fusion was abolished by restricting D2 in CqVCP in an ADP-bound state by NMS873 exposure in Hpt cells, followed by alkalization with chloroquine. In contrast to the control Hpt cells after viral fusion, rarely, accumulated endosomes containing WSSV were present in cells lacking CqVCP activity caused by preexposure to NMS873. (Left) Strong colocalization of endosomes and WSSV (arrows) in control cells without NMS873 exposure. (Right) Histogram analysis showing the fluorescence intensity of colocalization of RabGEF1 and VP28. (b) Increased autophagic activity induced by WSSV in a viral-dose-dependent manner in Hpt cells after blocking homotypic fusion of WSSV-containing vesicles and endosomes. The conversion of CqGABARAP-I to CqGABARAP-II occurred in a WSSV dose-dependent manner in Hpt cells after restricting D2 in CqVCP in an ADP-bound state by NMS873 exposure. Hpt cells were preexposed to NMS873, followed by WSSV infection at different MOI, as indicated. (C) Direction of WSSV to autophagosomes was inhibited by blocking homotypic fusion of endocytic vesicles and endosomes. (a) Restricting D2 in CqVCP in an ADP-bound state exposed to NMS873 did not inhibit conversion of CqGABARAP-I to CqGABARAP-II induced by exposure to AKTi 1/2. (b) Both formation of CqGABARAP puncta and acidic vesicles were substantially reduced by restricting D2 in CqVCP in an ADP-bound state by NMS873 exposure. Reduced formation of CqGABARAP puncta and acidic vesicles indicated inhibited fusion of endosomes and autophagosomes by blocking CqVCP activity with NMS873 exposure. (Top) CqGABARAP puncta immunostained with anti-GABARAP antibody are indicated by white arrows. (Bottom) Acidic vesicles stained by LysoTracker are indicated by green fluorescence. (c) Direction of WSSV to autophagosomes was inhibited by blocking homotypic fusion of endocytic vesicles and endosomes. (Left) WSSV virions strongly accumulated in autophagosomes (arrows) in control cells exposed solely to AKTi 1/2, but there was much less accumulation and small size in cells preexposed to NMS873, followed by AKTi 1/2 exposure. (Right) Histogram analysis showing significant inhibition of direction of WSSV to autophagosomes, as indicated by colocalized fluorescence intensity of VP28 and CqGABARAP. Hpt cells were preexposed to NMS873 for 2 h, followed by AKTi 1/2 exposure for 4 h, and infected with WSSV. (D) WSSV replication was profoundly inhibited by disruption of CqVCP-mediated homotypic fusion of endocytic vesicles and endosomes. Hpt cells were preexposed to NMS873, followed by WSSV infection. Both WSSV replication and translation were substantially inhibited by restricting D2 in CqVCP in an ADP-bound state by NMS873 exposure. Viral gene replication and protein synthesis were determined by qRT-PCR and Western blotting, respectively. (E) Propagation of progeny WSSV virions was strongly abolished by disrupting homotypic fusion of endocytic vesicles and endosomes mediated by CqVCP. (Top) Progeny virions were mostly present in the nuclear area in control Hpt cells 18 hpi but were rarely found in Hpt cells after blocking homotypic fusion by restricting D2 in CqVCP in an ADP-bound state by NMS873 exposure. (Left) Progeny virions are indicated by yellow arrows with immunostaining in control cells. Segregated endocytic WSSV virions or remaining viral components are indicated by white arrows in the cytoplasm of Hpt cells exposed to NMS873, in which the remaining viral components, such as VP28, were present only in the cytoplasmic area. (Right) Histogram analysis showing the relative percentages of Hpt cells containing progeny virions. Hpt cells containing progeny WSSV virions were relatively quantified by counting immunostaining signals of colocalization of VP28 and VP664 in cell nuclei. (Bottom) By TEM analysis, progeny virions were found only in control cells (yellow arrows), but not in NMS873-exposed Hpt cells, 18 hpi. Retained viral components were found to be present in endocytic vesicles in the cytoplasm. Homotypic fusion of endocytic vesicles and endosomes was disrupted by preexposure to NMS873 for 2 h in Hpt cells, followed by WSSV infection. N, nucleus; C, cytoplasm. Confocal microscopy analysis with WSSV infection was performed from 4 hpi, unless otherwise stated. n.s, no significant difference; *, P < 0.05; **, P < 0.01; ***, P < 0.001.

Journal: Journal of Virology

doi: 10.1128/JVI.01570-20

Figure Lengend Snippet: WSSV escaped from autophagic elimination via direction to the endosomal delivery system, substantially facilitated by CqVCP. (A) Most endocytic WSSV virions escaped from autophagy degradation via direction to endosomes, facilitated by CqVCP. (a) Dysfunctional endosomes, caused by blocking CqVCP ATPase activity, resulted in more WSSV aggregation than was caused by increasing autophagic activity. Aggregated WSSV virions were localized by immunostaining against viral envelope protein VP28 (white arrows). CqVCP ATPase activity was blocked by DBeQ exposure, and increased autophagic activity was induced by AKTi 1/2 exposure in Hpt cells. (b) Relative quantification of the integrated optical density of fluorescent VP28 signal (a, top row). (c) Relative quantification of colocalization of VP28 and VP664. Histogram analysis shows the integrated optical density of the colocalized fluorescent signals of VP28 and VP664 in Hpt cells (a, bottom row, yellow arrows). (d) Dysfunctional endosomes, caused by blocking CqVCP ATPase activity, resulted in more inhibition of WSSV replication than was caused by increasing autophagic activity. The relative gene expression of WSSV was determined by the presence of VP28 transcript 24 hpi in Hpt cells, using qRT-PCR. (B) Homotypic fusion of WSSV-containing endocytic vesicles and endosomes was significantly inhibited by restricting D2 in CqVCP in an ADP-bound state. (a) WSSV envelope fusion was profoundly inhibited by blocking homotypic fusion of WSSV-containing endocytic vesicles and endosomes. (Top) Inhibition of viral fusion was indirectly indicated by fluorescence of WSSV envelope protein VP28 (arrows). (Left) In contrast to control Hpt cells after viral fusion, much more fluorescent viral envelope protein, such as VP28, was present in cells lacking CqVCP activity caused by NMS873 exposure. (Right) Histogram analysis showing fluorescence intensity of intracellular viral envelope VP28 protein. (Bottom) Homotypic fusion was abolished by restricting D2 in CqVCP in an ADP-bound state by NMS873 exposure in Hpt cells, followed by alkalization with chloroquine. In contrast to the control Hpt cells after viral fusion, rarely, accumulated endosomes containing WSSV were present in cells lacking CqVCP activity caused by preexposure to NMS873. (Left) Strong colocalization of endosomes and WSSV (arrows) in control cells without NMS873 exposure. (Right) Histogram analysis showing the fluorescence intensity of colocalization of RabGEF1 and VP28. (b) Increased autophagic activity induced by WSSV in a viral-dose-dependent manner in Hpt cells after blocking homotypic fusion of WSSV-containing vesicles and endosomes. The conversion of CqGABARAP-I to CqGABARAP-II occurred in a WSSV dose-dependent manner in Hpt cells after restricting D2 in CqVCP in an ADP-bound state by NMS873 exposure. Hpt cells were preexposed to NMS873, followed by WSSV infection at different MOI, as indicated. (C) Direction of WSSV to autophagosomes was inhibited by blocking homotypic fusion of endocytic vesicles and endosomes. (a) Restricting D2 in CqVCP in an ADP-bound state exposed to NMS873 did not inhibit conversion of CqGABARAP-I to CqGABARAP-II induced by exposure to AKTi 1/2. (b) Both formation of CqGABARAP puncta and acidic vesicles were substantially reduced by restricting D2 in CqVCP in an ADP-bound state by NMS873 exposure. Reduced formation of CqGABARAP puncta and acidic vesicles indicated inhibited fusion of endosomes and autophagosomes by blocking CqVCP activity with NMS873 exposure. (Top) CqGABARAP puncta immunostained with anti-GABARAP antibody are indicated by white arrows. (Bottom) Acidic vesicles stained by LysoTracker are indicated by green fluorescence. (c) Direction of WSSV to autophagosomes was inhibited by blocking homotypic fusion of endocytic vesicles and endosomes. (Left) WSSV virions strongly accumulated in autophagosomes (arrows) in control cells exposed solely to AKTi 1/2, but there was much less accumulation and small size in cells preexposed to NMS873, followed by AKTi 1/2 exposure. (Right) Histogram analysis showing significant inhibition of direction of WSSV to autophagosomes, as indicated by colocalized fluorescence intensity of VP28 and CqGABARAP. Hpt cells were preexposed to NMS873 for 2 h, followed by AKTi 1/2 exposure for 4 h, and infected with WSSV. (D) WSSV replication was profoundly inhibited by disruption of CqVCP-mediated homotypic fusion of endocytic vesicles and endosomes. Hpt cells were preexposed to NMS873, followed by WSSV infection. Both WSSV replication and translation were substantially inhibited by restricting D2 in CqVCP in an ADP-bound state by NMS873 exposure. Viral gene replication and protein synthesis were determined by qRT-PCR and Western blotting, respectively. (E) Propagation of progeny WSSV virions was strongly abolished by disrupting homotypic fusion of endocytic vesicles and endosomes mediated by CqVCP. (Top) Progeny virions were mostly present in the nuclear area in control Hpt cells 18 hpi but were rarely found in Hpt cells after blocking homotypic fusion by restricting D2 in CqVCP in an ADP-bound state by NMS873 exposure. (Left) Progeny virions are indicated by yellow arrows with immunostaining in control cells. Segregated endocytic WSSV virions or remaining viral components are indicated by white arrows in the cytoplasm of Hpt cells exposed to NMS873, in which the remaining viral components, such as VP28, were present only in the cytoplasmic area. (Right) Histogram analysis showing the relative percentages of Hpt cells containing progeny virions. Hpt cells containing progeny WSSV virions were relatively quantified by counting immunostaining signals of colocalization of VP28 and VP664 in cell nuclei. (Bottom) By TEM analysis, progeny virions were found only in control cells (yellow arrows), but not in NMS873-exposed Hpt cells, 18 hpi. Retained viral components were found to be present in endocytic vesicles in the cytoplasm. Homotypic fusion of endocytic vesicles and endosomes was disrupted by preexposure to NMS873 for 2 h in Hpt cells, followed by WSSV infection. N, nucleus; C, cytoplasm. Confocal microscopy analysis with WSSV infection was performed from 4 hpi, unless otherwise stated. n.s, no significant difference; *, P < 0.05; **, P < 0.01; ***, P < 0.001.

Article Snippet: Rabbit anti-RabGEF1 polyclonal antibody was purchased from ABclonal (United States).

Techniques: Blocking Assay, Activity Assay, Immunostaining, Inhibition, Expressing, Quantitative RT-PCR, Fluorescence, Infection, Staining, Western Blot, Confocal Microscopy

Journal: eLife

Article Title: Endosomal Rab cycles regulate Parkin-mediated mitophagy

doi: 10.7554/eLife.31326

Figure Lengend Snippet: ( A ) HeLa cells transiently expressing mChery-Parkin and GFP-mRABGEF1 were treated with DMSO or valinomycin for 3 hr followed by immunostaining. The magnified pictures were shown in the right. Bars, 10 μm. ( B ) Total cell lysates of ( A ) were analyzed by immunoblotting. Anti-GFP antibody was used for the GFP-mRABGEF1 detection. * and # denote ubiquitinated forms and truncated forms, respectively. ( C ) Quantification of RABGEF1 recruitment to damaged mitochondria in ( A ). None, partial and complete denote that GFP-mRABGEF1 signals were overlapped with no, some of, and all mitochondria, respectively. ( D ) Recombinant ubiquitin (Ub) pre-treated with or without GST-TcPINK1 was subjected to pull-down assay with GST-mRABGEF1. W and E indicate wash and eluted fractions, respectively. 10%, 10% of input. ( E ) Percentages of the amount of ubiquitin in the eluted fraction in ( D ) were shown. The error bars represent mean ±SE from three independent experiments. ( F ) K48-linked and K63-linked Ub chains pre-treated with or without GST-TcPINK1 were subjected to pull-down assay with GST-mRABGEF1. ( G ) Interactions between GST-mRABGEF1 (WT or Y26A/A58D) and ubiquitin or phosphorylated ubiquitin were measured by ITC. N, stoichiometry of binding. 10.7554/eLife.31326.028 Figure 8—source data 1. Quantification of RABGEF1 recruitment to damaged mitochondria during mitophagy. 10.7554/eLife.31326.029 Figure 8—source data 2. Binding affinities of recombinant GST-mRABGEF1 with ubiquitin or phosphorylated ubiquitin. 10.7554/eLife.31326.030 Figure 8—source data 3. Binding affinities of recombinant GST-mRABGEF1 with ubiquitin or phosphorylated ubiquitin.

Article Snippet: The following antibodies were used for immunoblotting: rabbit anti-GFP (ab6556; Abcam, Cambridge, MA), mouse anti-MFN2 (ab56889; Abcam), rabbit anti-TOMM20 (sc-11415; Santa Cruz Biotechnology, Dallas, TX), rabbit anti-LC3B (L7543; Sigma, St. Louis, MO), mouse anti-MT-CO2 (ab110258; Abcam), mouse anti-Actin (MAB1501R; Millipore, Bedford, MA), mouse anti-RAB7 (ab50533; Abcam), rabbit anti- RABGEF1 (NBP1-49938; NOVUS BIOLOGICALS, Littleron, CO), mouse anti-CCZ1 (sc-514290; Santa Cruz Biotechnology), mouse anti-ubiquitin (sc-8017; Santa Cruz Biotechnology), and rabbit anti-S65 phosphorylated ubiquitin (described previously [ ]).

Techniques: Expressing, Immunostaining, Western Blot, Recombinant, Ubiquitin Proteomics, Pull Down Assay, Binding Assay

Journal: eLife

Article Title: Endosomal Rab cycles regulate Parkin-mediated mitophagy

doi: 10.7554/eLife.31326

Figure Lengend Snippet: ( A ) The indicated cells were treated with DMSO or valinomycin for 3 hr followed by immunostaining. Bars, 10 μm. Graphs for quantification of RABGEF1 recruitment to mitochondria were shown below the images. None, partial and complete denote that GFP-mRABGEF1 signals were overlapped with no, some of, and all mitochondria, respectively. The error bars represent mean ±SE and over 100 cells were counted in each of three separate wells. ( B ) WT and TBC1D15/17 DKO HCT116 cells stably expressing mCherry-Parkin and GFP-mRABGEF1 were treated with DMSO or valinomycin for 3 hr. GFP-mRABGEF1 signals were enhanced by immunostaining with anti-GFP antibody. Bars, 10 μm. ( C ) Total cell lysates in ( B ) were analyzed by immunoblotting. * and # denote ubiquitinated forms and truncated forms, respectively. 10.7554/eLife.31326.031 Figure 8—figure supplement 1—source data 1. This excel file contains quantification of RABGEF1 (WT and Y26A/A58D mutant) recruitment to mitochondria in HCT116 (WT and TBC1D15/17 DKO) cells.

Techniques: Immunostaining, Stable Transfection, Expressing, Western Blot, Mutagenesis

Journal: eLife

Article Title: Endosomal Rab cycles regulate Parkin-mediated mitophagy

doi: 10.7554/eLife.31326

Figure Lengend Snippet: ( A ) GFP-mRABGEF1 was transiently expressed in siRNA-treated HeLa cells. The cells were then treated with valinomycin for 3 hr followed by immunostaining. Bars, 20 μm. ( B ) Quantification of mitochondrial recruitment of GFP-mRABGEF1 in HeLa cells. ( C ) Quantification of mitochondrial recruitment of GFP-mRABGEF1 in HCT116 cells. None, partial and complete denote that GFP-mRABGEF1 signals were overlapped with no, some of, and all mitochondria, respectively. The error bars represent mean ± SE and over 100 cells were counted in each of three separate wells. 10.7554/eLife.31326.032 Figure 8—figure supplement 2—source data 2. Quantification of RABGEF1 recruitment to mitochondria in HeLa cells treated with the indicated siRNA during mitophagy. 10.7554/eLife.31326.033 Figure 8—figure supplement 2—source data 3. Quantification of RABGEF1 recruitment to mitochondria in HCT116 cells treated with the indicated siRNA during mitophagy.

Techniques: Immunostaining

Journal: eLife

Article Title: Endosomal Rab cycles regulate Parkin-mediated mitophagy

doi: 10.7554/eLife.31326

Figure Lengend Snippet: ( A ) WT and RABGEF1-mAID HCT116 cells were treated with or without IAA for 16 hr. Total cell lysates were analyzed by immunoblotting. ( B ) Quantification of Parkin recruitment to mitochondria in WT and RABGEF1-mAID HCT116 cells after 3 hr of valinomycin treatment. Partial and complete denote that YFP-Parkin signals were overlapped with some of and all mitochondria, respectively. ( C ) YFP-Parkin stably expressing WT and RABGEF1-mAID HCT116 cells pre-treated with IAA were treated with valinomycin for the indicated times. Total cell lysates were analyzed by immunoblotting. ( D ) WT and RABGEF1-mAID HCT116 cells stably expressing YFP-Parkin and mt-mKeima were treated with IAA for 16 hr followed by DMSO or OAQ for 6 hr and subjected to FACS analysis. Plots are representative of n = 3 experiments. ( E ) Quantification of mitophagy in ( D ). Error bars represent mean ±SE of three independent experiments. Statistical differences were determined by student’s t-test. *p<0.05. 10.7554/eLife.31326.035 Figure 9—source data 1. Quantification of YFP-Parkin recruitment to mitochondria in RABGEF1-mAID HCT116 and the corresponding WT cells during mitophagy. 10.7554/eLife.31326.036 Figure 9—source data 2. Quantification of mitophagy using mt-mKeima and FACS analysis.

Techniques: Western Blot, Stable Transfection, Expressing

Journal: eLife

Article Title: Endosomal Rab cycles regulate Parkin-mediated mitophagy

doi: 10.7554/eLife.31326

Figure Lengend Snippet:

Techniques: Sequencing, Ubiquitin Proteomics, Protease Inhibitor, Western Blot, Recombinant, Software, Microscopy

Journal: eLife

Article Title: Endosomal Rab cycles regulate Parkin-mediated mitophagy

doi: 10.7554/eLife.31326

Figure Lengend Snippet:

Article Snippet: Antibody , Rabbit anti-RABGEF1 (polyclonal) , NOVUS BIOLOGICALS , NBP1-49938 AB_10012128 , 1:500 (WB).

Techniques: Sequencing, Ubiquitin Proteomics, Protease Inhibitor, Western Blot, Recombinant, Software, Microscopy

Review

Structured Review

Images

1) Product Images from "Endosomal Rab cycles regulate Parkin-mediated mitophagy"

Similar Products

Image Search Results