Journal: bioRxiv

Article Title: System-wide profiling reveals metabolic alterations in a G2019S LRRK2 knockin mouse model of Parkinson’s disease: systemic depletion of pyrimidine nucleosides

doi: 10.64898/2025.12.31.697225

Figure Lengend Snippet: a Representative image of TH immunostaining in the substantia nigra. Scale bar: 500 µm. Quantification of TH-positive neurons ( b ) and Nissl-positive neurons ( c ) in the substantia nigra ( n = 3-7 per group). d Representative images of TH immunostaining in the striatum. Scale bar: 100 µm. e Quantification of TH-positive area in the striatum ( n = 5-6 per group). f Relative striatal dopamine level measured by LC-MS ( n = 5-6 per group). Western blot analysis ( g-h ) and quantification ( i-n ) of LRRK2 and Rab12 expression levels in the striatum ( g, i-k ) and ventral midbrain ( h, l-n ) of G2019S LRRK2 knockin and wild-type mice under control (Con.) diet and HFD conditions ( n = 3 per group). Data are presented as mean ± SEM. A two-way ANOVA was performed to evaluate the effects of high-fat diet and G2019S LRRK2 mutant and their interaction followed by Fisher’s LSD comparisons. WT/WT, wild-type mice; KI/KI, homozygous G2019S LRRK2 knockin mice. Data are presented as mean ± SEM. A two-way ANOVA was performed to evaluate the effects of high-fat diet and G2019S LRRK2 mutant and their interaction followed by Fisher’s LSD comparisons. Significant main effects were found (* P <0.05, ** P <0.01, *** P <0.001).

Article Snippet: Primary antibodies used for Western blotting include: LRRK2 (75-253, NeuroMab), pS935-LRRK2 (ab133450, Abcam), pS1292-LRRK2 (ab203181, Abcam), Rab8 (D22D8, Cell Signaling Technology), pT72-Rab8a (ab230260, Abcam), Rab10 (8127S; Cell Signaling Technology), pThr73-Rab10 (ab230261, Abcam), Rab12 (18843-1-AP, Protein Tech), pSer106-Rab12 (ab256487, Abcam), Actin (MAB1501, Millipore), Thymidine Phosphorylase (AF7568, Novus Biologicals).

Techniques: Immunostaining, Liquid Chromatography with Mass Spectroscopy, Western Blot, Expressing, Knock-In, Control, Mutagenesis

Journal: bioRxiv

Article Title: System-wide profiling reveals metabolic alterations in a G2019S LRRK2 knockin mouse model of Parkinson’s disease: systemic depletion of pyrimidine nucleosides

doi: 10.64898/2025.12.31.697225

Figure Lengend Snippet: a Diagram showing that thymidine phosphorylase catalyzes the conversion of thymidine and deoxyuridine into their respective bases (thymine and uracil) and 2-deoxyribose 1-phosphate. b Western blot analysis of thymidine phosphorylase (TP) expression levels in brain and liver in 5 month-old G2019S LRRK2 knockin mice and controls ( n = 2 per group). c qRT-PCR analysis of TYMP levels in ventral midbrain of 5 month-old G2019S LRRK2 knockin mice and controls ( n = 4 per group). Data are presented as mean ± SEM. Unpaired two-tailed Student’s t -test was performed to evaluate the effects of G2019S LRRK2 mutation, no significant effect was found. d Volcano plot showing differential gene expression in the liver of 5 month-old G2019S LRRK2 knockin mice compared to wild-type controls. Each point represents one gene. X-axis shows log 2 (fold change) in gene expression in G2019S LRRK2 knockin mice compared to wild-type controls. Y-axis shows -log 10 (adjusted p -value) from limma’s empirical Bayes moderated analysis (eBayes) combined with Benjamini-Hochberg (BH) for multiple testing correction ( n = 4 per group). Gene expression with adjusted P <0.05 are considered significantly altered. Significantly upregulated genes are highlighted in red and significantly downregulated genes are highlighted in blue. e Volcano plot illustrating enriched biological processes in G2019S LRRK2 knockin mice compared to wild-type controls as identified by gene set enrichment analysis (GSEA). Each point represents one process. X-axis shows normalized enrichment score, Y-axis shows -log 10 (adjusted p -value), significance was defined as a Benjamini–Hochberg adjusted P <0.05 ( n = 4 per group). Significantly upregulated pathways are highlighted in red and significantly downregulated pathways are highlighted in blue. f Western blot analysis and quantification ( g-m ) of TP, total and phosphorylated LRRK2, Rab10, and Rab12 expression levels in the liver of G2019S LRRK2 knockin and wild-type mice under Con.diet and HFD conditions ( n = 3 per group). WT/WT, wild-type mice; KI/KI, homozygous G2019S LRRK2 knockin mice. Data are presented as mean ± SEM. A two-way ANOVA was performed to evaluate the effects of high-fat diet and G2019S LRRK2 mutant and their interaction followed by Fisher’s LSD comparisons. Significant main effects were found (* P <0.05, ** P <0.01).

Article Snippet: Primary antibodies used for Western blotting include: LRRK2 (75-253, NeuroMab), pS935-LRRK2 (ab133450, Abcam), pS1292-LRRK2 (ab203181, Abcam), Rab8 (D22D8, Cell Signaling Technology), pT72-Rab8a (ab230260, Abcam), Rab10 (8127S; Cell Signaling Technology), pThr73-Rab10 (ab230261, Abcam), Rab12 (18843-1-AP, Protein Tech), pSer106-Rab12 (ab256487, Abcam), Actin (MAB1501, Millipore), Thymidine Phosphorylase (AF7568, Novus Biologicals).

Techniques: Western Blot, Expressing, Knock-In, Quantitative RT-PCR, Two Tailed Test, Mutagenesis, Gene Expression

Journal: bioRxiv

Article Title: System-wide profiling reveals metabolic alterations in a G2019S LRRK2 knockin mouse model of Parkinson’s disease: systemic depletion of pyrimidine nucleosides

doi: 10.64898/2025.12.31.697225

Figure Lengend Snippet: a Schematic diagram showing central energy and biosynthetic pathways involving glucose and fatty acids. b-p Bar plot showing comparation of selected metabolites (relative abundance) across groups ( n = 6 per group). These metabolites reflect alterations in glycolysis ( b-c ), TCA cycle ( d-e ), pentose phosphate pathway ( f-j ), and nucleotide metabolism ( k-p ). Data are presented as mean ± SEM in bar plot. A two-way ANOVA was performed to evaluate the effects of high-fat diet and G2019S LRRK2 and their interaction, Tukey’s post hoc test was used for multiple comparisons. Significant main and interaction effects were found (* P <0.05, ** P <0.01, *** P <0.001, **** P <0.0001). q-s Representative images of H&E staining ( q ), CD68 ( r ) and CD3 ( s ) immunostaining of the lungs, scale bar:100 µm. t-w Volcano plot showing differences in the abundance of proteins between HFD and Con. diet groups in wild-type mice ( t ) and G2019S LRRK2 knockin mice ( u ); between G2019S LRRK2 knockin and wild-type mice under Con. diet ( v ) and HFD ( w ) conditions ( n = 4 per group). Each point represents a protein. X-axis shows log 2 (fold change) in protein abundance between each comparations. Y-axis shows -log 10 (adjusted p -value) from limma’s empirical Bayes moderated analysis (eBayes) combined with Benjamini-Hochberg (BH) for multiple testing correction ( n = 4 per group). Proteins with adjusted P <0.05 are considered significantly altered. Significantly upregulated proteins are highlighted in red and significantly downregulated proteins are highlighted in blue. x Western blot analysis and quantification ( y-ae ) of total and phosphorated LRRK2, Rab8a, Rab10 and Rab12 expression levels in the lung of G2019S LRRK2 knockin and wild-type mice under Con.diet and HFD conditions ( n = 3 per group). WT/WT, wild-type mice; KI/KI, homozygous G2019S LRRK2 knockin mice. Data are presented as mean ± SEM. A two-way ANOVA was performed to evaluate the effects of high-fat diet and G2019S LRRK2 and their interaction by Fisher’s LSD comparisons. Significant main and interaction effects were found (* P <0.05, ** P <0.01, *** P <0.001, **** P <0.0001).

Article Snippet: Primary antibodies used for Western blotting include: LRRK2 (75-253, NeuroMab), pS935-LRRK2 (ab133450, Abcam), pS1292-LRRK2 (ab203181, Abcam), Rab8 (D22D8, Cell Signaling Technology), pT72-Rab8a (ab230260, Abcam), Rab10 (8127S; Cell Signaling Technology), pThr73-Rab10 (ab230261, Abcam), Rab12 (18843-1-AP, Protein Tech), pSer106-Rab12 (ab256487, Abcam), Actin (MAB1501, Millipore), Thymidine Phosphorylase (AF7568, Novus Biologicals).

Techniques: Staining, Immunostaining, Knock-In, Quantitative Proteomics, Western Blot, Expressing

Journal: bioRxiv

Article Title: System-wide profiling reveals metabolic alterations in a G2019S LRRK2 knockin mouse model of Parkinson’s disease: systemic depletion of pyrimidine nucleosides

doi: 10.64898/2025.12.31.697225

Figure Lengend Snippet: a Diagram showing that thymidine phosphorylase catalyzes the conversion of thymidine and deoxyuridine into their respective bases (thymine and uracil) and 2-deoxyribose 1-phosphate. b Western blot analysis of thymidine phosphorylase (TP) expression levels in brain and liver in 5 month-old G2019S LRRK2 knockin mice and controls ( n = 2 per group). c qRT-PCR analysis of TYMP levels in ventral midbrain of 5 month-old G2019S LRRK2 knockin mice and controls ( n = 4 per group). Data are presented as mean ± SEM. Unpaired two-tailed Student’s t -test was performed to evaluate the effects of G2019S LRRK2 mutation, no significant effect was found. d Volcano plot showing differential gene expression in the liver of 5 month-old G2019S LRRK2 knockin mice compared to wild-type controls. Each point represents one gene. X-axis shows log 2 (fold change) in gene expression in G2019S LRRK2 knockin mice compared to wild-type controls. Y-axis shows -log 10 (adjusted p -value) from limma’s empirical Bayes moderated analysis (eBayes) combined with Benjamini-Hochberg (BH) for multiple testing correction ( n = 4 per group). Gene expression with adjusted P <0.05 are considered significantly altered. Significantly upregulated genes are highlighted in red and significantly downregulated genes are highlighted in blue. e Volcano plot illustrating enriched biological processes in G2019S LRRK2 knockin mice compared to wild-type controls as identified by gene set enrichment analysis (GSEA). Each point represents one process. X-axis shows normalized enrichment score, Y-axis shows -log 10 (adjusted p -value), significance was defined as a Benjamini–Hochberg adjusted P <0.05 ( n = 4 per group). Significantly upregulated pathways are highlighted in red and significantly downregulated pathways are highlighted in blue. f Western blot analysis and quantification ( g-m ) of TP, total and phosphorylated LRRK2, Rab10, and Rab12 expression levels in the liver of G2019S LRRK2 knockin and wild-type mice under Con.diet and HFD conditions ( n = 3 per group). WT/WT, wild-type mice; KI/KI, homozygous G2019S LRRK2 knockin mice. Data are presented as mean ± SEM. A two-way ANOVA was performed to evaluate the effects of high-fat diet and G2019S LRRK2 mutant and their interaction followed by Fisher’s LSD comparisons. Significant main effects were found (* P <0.05, ** P <0.01).

Article Snippet: Primary antibodies used for Western blotting include: LRRK2 (75-253, NeuroMab), pS935-LRRK2 (ab133450, Abcam), pS1292-LRRK2 (ab203181, Abcam), Rab8 (D22D8, Cell Signaling Technology), pT72-Rab8a (ab230260, Abcam), Rab10 (8127S; Cell Signaling Technology), pThr73-Rab10 (ab230261, Abcam), Rab12 (18843-1-AP, Protein Tech), pSer106-Rab12 (ab256487, Abcam), Actin (MAB1501, Millipore), Thymidine Phosphorylase (AF7568, Novus Biologicals).

Techniques: Western Blot, Expressing, Knock-In, Quantitative RT-PCR, Two Tailed Test, Mutagenesis, Gene Expression

Journal: bioRxiv

Article Title: System-wide profiling reveals metabolic alterations in a G2019S LRRK2 knockin mouse model of Parkinson’s disease: systemic depletion of pyrimidine nucleosides

doi: 10.64898/2025.12.31.697225

Figure Lengend Snippet: a Schematic diagram showing central energy and biosynthetic pathways involving glucose and fatty acids. b-p Bar plot showing comparation of selected metabolites (relative abundance) across groups ( n = 6 per group). These metabolites reflect alterations in glycolysis ( b-c ), TCA cycle ( d-e ), pentose phosphate pathway ( f-j ), and nucleotide metabolism ( k-p ). Data are presented as mean ± SEM in bar plot. A two-way ANOVA was performed to evaluate the effects of high-fat diet and G2019S LRRK2 and their interaction, Tukey’s post hoc test was used for multiple comparisons. Significant main and interaction effects were found (* P <0.05, ** P <0.01, *** P <0.001, **** P <0.0001). q-s Representative images of H&E staining ( q ), CD68 ( r ) and CD3 ( s ) immunostaining of the lungs, scale bar:100 µm. t-w Volcano plot showing differences in the abundance of proteins between HFD and Con. diet groups in wild-type mice ( t ) and G2019S LRRK2 knockin mice ( u ); between G2019S LRRK2 knockin and wild-type mice under Con. diet ( v ) and HFD ( w ) conditions ( n = 4 per group). Each point represents a protein. X-axis shows log 2 (fold change) in protein abundance between each comparations. Y-axis shows -log 10 (adjusted p -value) from limma’s empirical Bayes moderated analysis (eBayes) combined with Benjamini-Hochberg (BH) for multiple testing correction ( n = 4 per group). Proteins with adjusted P <0.05 are considered significantly altered. Significantly upregulated proteins are highlighted in red and significantly downregulated proteins are highlighted in blue. x Western blot analysis and quantification ( y-ae ) of total and phosphorated LRRK2, Rab8a, Rab10 and Rab12 expression levels in the lung of G2019S LRRK2 knockin and wild-type mice under Con.diet and HFD conditions ( n = 3 per group). WT/WT, wild-type mice; KI/KI, homozygous G2019S LRRK2 knockin mice. Data are presented as mean ± SEM. A two-way ANOVA was performed to evaluate the effects of high-fat diet and G2019S LRRK2 and their interaction by Fisher’s LSD comparisons. Significant main and interaction effects were found (* P <0.05, ** P <0.01, *** P <0.001, **** P <0.0001).

Article Snippet: Primary antibodies used for Western blotting include: LRRK2 (75-253, NeuroMab), pS935-LRRK2 (ab133450, Abcam), pS1292-LRRK2 (ab203181, Abcam), Rab8 (D22D8, Cell Signaling Technology), pT72-Rab8a (ab230260, Abcam), Rab10 (8127S; Cell Signaling Technology), pThr73-Rab10 (ab230261, Abcam), Rab12 (18843-1-AP, Protein Tech), pSer106-Rab12 (ab256487, Abcam), Actin (MAB1501, Millipore), Thymidine Phosphorylase (AF7568, Novus Biologicals).

Techniques: Staining, Immunostaining, Knock-In, Quantitative Proteomics, Western Blot, Expressing

Journal: Nature Communications

Article Title: Rab41-mediated ESCRT machinery repairs membrane rupture by a bacterial toxin in xenophagy

doi: 10.1038/s41467-023-42039-2

Figure Lengend Snippet: a , b Screening for Rab GTPases that affect intracellular bacterial viability. HeLa wild-type (WT) ( a ) or ATG5 -knockout (KO) ( b ) cells transfected with the indicated siRNA were infected with GAS. siNT means non-targeting control siRNA. Colony counting (determination of colony forming unit, CFU) was conducted to determine the number of invading and surviving GAS; the bacterial survival data were calculated as the ratio of “intracellular live GAS at 6 h” to “total intracellular GAS at 2 h”. Data are represented as individual values (circles) and mean ± standard error of the mean (SEM) ( n = 3 biologically independent experiments). One-way analysis of variance (ANOVA), Dunnett’s test. Red P values indicated a significant increase and blue ones means a significant decrease. c – e HeLa cells stably expressing GFP-LC3 were transfected with the indicated siRNA, and infected with GAS for 4 h, then fixed and immunostained for endogenous LAMP1 (red). Cellular and bacterial DNA were stained with DAPI (cyan). c Shown representative microscopic images are single slice. Scale bar, 10 μm. ( d ) GcAV formation was quantified as the percentages of cells with GcAVs. Data are represented as individual values and mean ± SEM of more than three independent experiments (200

Article Snippet: For immunoblot analysis, the following antibodies were used as a primary antibody: streptolysin O (SLO) (ab188539; Abcam, 1:1000), HA (HA124; Nacalai Tesque, 1:1000), FLAG (M2; A2220; Sigma-Aldrich, 1:1000), GFP (GF200; 04363-24; Nacalai Tesque, 1:1000), MBP (E8032; New England BioLabs, 1:1000), GAPDH (sc-47724; Santa cruz biotechnology), Rab8A (D22D8; 6975 S; Cell Signaling Technology, 1:1000), Rab41 (NBP2-83434; NOVUS Biologicals, 1:1000), Rab41 (18818-1-AP; Proteintech Group, 1:500), VPS4A/B (17673-1-AP, Proteintech Group, 1:1000), and TOM1L2 (GTX106295; GENETEX, 1:1000).

Techniques: Knock-Out, Transfection, Infection, Control, Stable Transfection, Expressing, Staining

Journal: Nature Communications

Article Title: Rab41-mediated ESCRT machinery repairs membrane rupture by a bacterial toxin in xenophagy

doi: 10.1038/s41467-023-42039-2

Figure Lengend Snippet: a Localization of the ESCRT-III component CHMP4B during GAS infection. HeLa cells transfected with CHMP4B-EGFP and mCherry-LC3 were infected with GAS for 4 h, fixed, then stained with DAPI. Shown are representative confocal single slice images of three independent experiments. Scale bar, 2 μm. b HeLa cells were infected with GAS for 4 h, fixed, then immunostained for endogenous LC3 and CHMP4B. Cellular and bacterial DNA were stained with DAPI. Shown confocal images are single slice and representative of three independent experiments. Scale bar, 2 μm. c HeLa cells stably expressing GFP-LC3 were infected with GAS for 4 h, fixed, then immunostained for endogenous CHMP6, VPS28, or SNF8. Cellular and bacterial DNA were stained with DAPI. Shown confocal images are single slice and representative of three independent experiments. Scale bar, 10 μm. d HeLa cells stably expressing GFP-LC3 were infected with GAS in absence or presence of BAPTA-AM (10 μM) for 4 h, fixed, then immunostained for endogenous CHMP4B. Cellular and bacterial DNA were stained with DAPI. Representative confocal single slice images and quantification of CHMP4B recruitment to GcAVs. Scale bar, 10 μm. The proportion of the LC3 signal (GcAVs) overlapping the CHMP4B signal were quantified by Mander’s coefficient M1. Data are individual values and mean ± SEM ( n = 50 GcAVs examined over three independent experiments). Unpaired two-tailed t test. e – g HeLa cells stably expressing GFP-LC3 were infected with GAS for the indicated time, fixed, then immunostained for endogenous LAMP1 and CHMP4B. Cellular and bacterial DNA were stained with DAPI. Representative confocal micrographs of single slice ( e ) and the proportion of the LC3 signal (GcAVs) overlapping the LAMP1 or CHMP4B signal were quantified by Mander’s coefficient M1 ( f ). Scale bar, 2 μm. Data are mean (circles) ± SEM (shadows) ( n = 20 GcAVs examined in each three independent experiments). The percentage of GcAV-positive cells for LAMP1 and CHMP4B were counted using microscopy ( g ). h – j HeLa cells stably expressing GFP-LC3 were transfected with the indicated siRNA, and infected with GAS. Cells were fixed, immunostained for endogenous CHMP4B and stained with DAPI. Representative confocal micrographs of single slice ( h ), and the proportion of the LC3 signal (GcAVs) overlapping CHMP4B was quantified by Mander’s coefficient M1 ( i , j ). Scale bar, 10 μm. Data in ( i , j ) are individual values and the mean ± SEM (30 GcAVs examined over three independent experiments). One-way ANOVA, Dunnett’s test. k , l HeLa WT, Rab8A KO, Rab41 KO cells stably expressing GFP-LC3 were infected with GAS for 4 h, fixed, and immunostained for endogenous CHMP4B. Representative confocal images of single slice ( k ) and the proportion of LC3 signal (GcAVs) overlapping CHMP4B quantified by Mander’s coefficient M1 ( l ). Scale bar, 10 μm. Data in ( l ) are individual values and the mean ± SEM (30 GcAVs examined over three independent experiments). One-way ANOVA, Dunnett’s test. Source data are provided as a Source Data file.

Article Snippet: For immunoblot analysis, the following antibodies were used as a primary antibody: streptolysin O (SLO) (ab188539; Abcam, 1:1000), HA (HA124; Nacalai Tesque, 1:1000), FLAG (M2; A2220; Sigma-Aldrich, 1:1000), GFP (GF200; 04363-24; Nacalai Tesque, 1:1000), MBP (E8032; New England BioLabs, 1:1000), GAPDH (sc-47724; Santa cruz biotechnology), Rab8A (D22D8; 6975 S; Cell Signaling Technology, 1:1000), Rab41 (NBP2-83434; NOVUS Biologicals, 1:1000), Rab41 (18818-1-AP; Proteintech Group, 1:500), VPS4A/B (17673-1-AP, Proteintech Group, 1:1000), and TOM1L2 (GTX106295; GENETEX, 1:1000).

Techniques: Infection, Transfection, Staining, Stable Transfection, Expressing, Two Tailed Test, Microscopy

Journal: Nature Communications

Article Title: Rab41-mediated ESCRT machinery repairs membrane rupture by a bacterial toxin in xenophagy

doi: 10.1038/s41467-023-42039-2

Figure Lengend Snippet: a Co-immunoprecipitation between FLAG-Rab41 and GFP-ESCRT proteins during GAS infection. HeLa cells expressing GFP tagged ESCRT components and FLAG-Rab41 were infected with GAS for 4 h. Cell extract were immunoprecipitated (IP) with GFP-Trap beads. The resulting samples, IP and whole cell lysate (WCL), were analyzed by immunoblot analysis. Data shown are representative of three independent experiments. b , c The recruitment of ESCRT-I subunit VPS28 to GcAVs in Rab41 -knockout cells. HeLa WT and Rab41 knockout (KO) cells were infected with GAS for 4 h, fixed, and immunostained for endogenous VPS28. Cellular and bacterial DNA were stained with DAPI. The proportion of the LC3 vacuole signal (GcAVs) overlapping VPS28 was quantified by Mander’s coefficient M1. Representative confocal images of single slice ( b ) and quantification of VPS4 recruitment to GcAVs ( c ). Data are individual values and mean ± SEM ( n = 30 GcAVs examined over three independent experiments). Unpaired two-tailed t test. Scale bar, 10 μm. d , e The recruitment of VPS4 to GcAVs in Rab41 -knockout cells. HeLa WT and Rab41 knockout cells expressing FLAG-Rab41, -Rab41 T44N, or -Q89L were infected with GAS for 4 h, fixed, and immunostained for endogenous VPS4. Cellular and bacterial DNA were stained with DAPI. The proportion of the LC3 vacuole signal (GcAVs) overlapping VPS4 was quantified by Mander’s coefficient M1. Representative confocal images of single slice ( d ) and quantification of VPS4 recruitment to GcAVs ( e ). Scale bar, 10 μm. Data are individual values and mean ± SEM ( n = 30 GcAVs examined over three independent experiments). One-way ANOVA, Tukey’s test. f HeLa WT and Rab41 knockout cells expressing FLAG-Rab41, -Rab41 T44N, or -Q89L were infected with GAS JRS4 and the intracellular bacterial CFU was determined at 6 hpi. Data are individual values and mean ± SEM ( n = 3 biologically independent experiments). One-way ANOVA, Tukey’s test. g Immunoprecipitation analysis. HeLa cells were infected with GAS for 4 h and then lysed with IP lysis buffer. Cell lysate were immunoprecipitated with anti-Rab41 or control rabbit antibody. The resulting samples were analyzed by immunoblot analysis. Data shown are representative of three independent experiments. h Domain organization and deletion mutants of VPS4. i , j Co-immunoprecipitation analysis. HEK293T were transfected with GFP tagged VPS4 deletion mutants and FLAG tagged Rab41, and then cell lysates were immunoprecipitated with GFP-Trap beads. The resulting samples were analyzed by immunoblot analysis. Data shown are representative of three independent experiments. k HeLa cells transfected with VPS4-EGFP FL, MIT or LA domain and mCherry-LC3 were infected with GAS for 4 h, fixed and stained with DAPI. Shown are representative confocal single-slice images of three independent experiments. Scale bar, 10 μm. l Co-immunoprecipitation analysis. HEK293T were transfected with GFP tagged Rab41 wild-type or Rab41 S94A mutant, and then cell lysates were immunoprecipitated with GFP-Trap beads. The resulting samples were analyzed by immunoblot analysis. Data shown are representative of three independent experiments. m HeLa WT and Rab41 knockout cells expressing GFP-Rab41 constructs were infected with GAS, and intracellular survival bacteria were determined by colony counting. Data are individual values and mean ± SEM ( n = 3 biologically independent experiments). One-way ANOVA, Tukey’s test. Source data are provided as a Source Data file.

Article Snippet: For immunoblot analysis, the following antibodies were used as a primary antibody: streptolysin O (SLO) (ab188539; Abcam, 1:1000), HA (HA124; Nacalai Tesque, 1:1000), FLAG (M2; A2220; Sigma-Aldrich, 1:1000), GFP (GF200; 04363-24; Nacalai Tesque, 1:1000), MBP (E8032; New England BioLabs, 1:1000), GAPDH (sc-47724; Santa cruz biotechnology), Rab8A (D22D8; 6975 S; Cell Signaling Technology, 1:1000), Rab41 (NBP2-83434; NOVUS Biologicals, 1:1000), Rab41 (18818-1-AP; Proteintech Group, 1:500), VPS4A/B (17673-1-AP, Proteintech Group, 1:1000), and TOM1L2 (GTX106295; GENETEX, 1:1000).

Techniques: Immunoprecipitation, Infection, Expressing, Western Blot, Knock-Out, Staining, Two Tailed Test, Lysis, Control, Transfection, Mutagenesis, Construct, Bacteria

Journal: Nature Communications

Article Title: Rab41-mediated ESCRT machinery repairs membrane rupture by a bacterial toxin in xenophagy

doi: 10.1038/s41467-023-42039-2

Figure Lengend Snippet: a , b Rab41 recruitment to GcAVs. HeLa cells expressing EmGFP-Rab41 and mCherry-LC3 were infected with GAS for 4 h, fixed, and stained with DAPI. ( a ) Representative confocal single slice images of EmGFP-Rab41 localization in uninfected or GAS-infected cells and ( b ) the time course of Rab41 and CHMP4B recruitment to GcAVs. Data in ( b ) represent are mean (circles) ± SEM (shadow) from three independent experiments and 50 GcAVs

Article Snippet: For immunoblot analysis, the following antibodies were used as a primary antibody: streptolysin O (SLO) (ab188539; Abcam, 1:1000), HA (HA124; Nacalai Tesque, 1:1000), FLAG (M2; A2220; Sigma-Aldrich, 1:1000), GFP (GF200; 04363-24; Nacalai Tesque, 1:1000), MBP (E8032; New England BioLabs, 1:1000), GAPDH (sc-47724; Santa cruz biotechnology), Rab8A (D22D8; 6975 S; Cell Signaling Technology, 1:1000), Rab41 (NBP2-83434; NOVUS Biologicals, 1:1000), Rab41 (18818-1-AP; Proteintech Group, 1:500), VPS4A/B (17673-1-AP, Proteintech Group, 1:1000), and TOM1L2 (GTX106295; GENETEX, 1:1000).

Techniques: Expressing, Infection, Staining, Knock-In, Knock-Out, Incubation, Two Tailed Test

Journal: Nature Communications

Article Title: Rab41-mediated ESCRT machinery repairs membrane rupture by a bacterial toxin in xenophagy

doi: 10.1038/s41467-023-42039-2

Figure Lengend Snippet: a – c HeLa cells transfected with indicated siRNA were treated with LLOMe and fixed at indicated time points, and immunostained for endogenous CHMP4B. Representative confocal single-slice images are shown ( a ). Enlarged CHMP4B-positive vacuoles are indicated by yellow arrows. Scale bar, 10 μm. The numbers of CHMP4B puncta were quantified from randomly-selected cells using imageJ, and data are individual values and mean ± SEM ( n = 50 GcAVs examined over three independent experiments). One-way ANOVA, Tukey’s test. d HeLa cells stably expressing GFP-LC3 were treated with LLOMe for 1 h, fixed, and then immunostained for LAMP1 and CHMP4B. Shown are representative confocal single-slice images of three independent experiments. Scale bar, 10 μm. e HeLa WT or Rab41 knockout cells stably expressing GFP-LC3 were treated with LLOMe for 1 h, fixed, and then immunostained for CHMP4B or VPS4. Shown are representative confocal single-slice images of three independent experiments. Fluorescent line scans across the yellow dashed line in the merged insets. Scale bar, 10 μm. f Schematic depicting Rab41-independent lysosomal repair and -dependent autolysosomal repair. Source data are provided as a Source Data file.

Article Snippet: For immunoblot analysis, the following antibodies were used as a primary antibody: streptolysin O (SLO) (ab188539; Abcam, 1:1000), HA (HA124; Nacalai Tesque, 1:1000), FLAG (M2; A2220; Sigma-Aldrich, 1:1000), GFP (GF200; 04363-24; Nacalai Tesque, 1:1000), MBP (E8032; New England BioLabs, 1:1000), GAPDH (sc-47724; Santa cruz biotechnology), Rab8A (D22D8; 6975 S; Cell Signaling Technology, 1:1000), Rab41 (NBP2-83434; NOVUS Biologicals, 1:1000), Rab41 (18818-1-AP; Proteintech Group, 1:500), VPS4A/B (17673-1-AP, Proteintech Group, 1:1000), and TOM1L2 (GTX106295; GENETEX, 1:1000).

Techniques: Transfection, Stable Transfection, Expressing, Knock-Out

Journal: Nature Communications

Article Title: Rab41-mediated ESCRT machinery repairs membrane rupture by a bacterial toxin in xenophagy

doi: 10.1038/s41467-023-42039-2

Figure Lengend Snippet: a Volcano plot showing the subset of proteins with enriched in GFP-Rab41 precipitates. The red and blue plots show proteins that are twofold higher or lower abundance in the LLOMe treatment relative to the DMSO treatment, respectively, with the significance of enrichment (−log10 of adjusted P value) ( n = 4 biological replicates). HEK293T cells expressing GFP or GFP-Rab41 were treated with LLOMe and GFP or GFP-Rab41 was precipitated with GFP-Trap beads, and precipitated proteins were identified by mass spectrometry. Unpaired two-tailed t test. b HeLa cells expressing EmGFP-TOM1L2 and mCherry-LGALS3 were treated with 1 mM of LLOMe for 30 min. Shown are representative confocal single-slice images of three independent experiments. Scale bar, 10 μm. c HeLa cells expressing EmGFP-TOM1L2 and mCherry-LC3 or expressing EmGFP-LC3 were infected with GAS for 4 h, and endogenous TOM1L2 was stained with an anti-TOM1L2 antibody. Shown are representative confocal single-slice images of three independent experiments. Cellular and bacterial DNA were stained with DAPI. Scale bar, 10 μm. d Co-immunoprecipitation assay. HeLa cells were incubated in the presence or absence of LLOMe for 1 h and cell lysates were immunoprecipitated with anti-Rab41 antibody. The resulting samples were analyzed by immunoblot. Data shown are representative of three independent experiments. e HeLa cells expressing EmGFP-Rab41 and FLAG-TOM1L2 were infected with GAS for 4 h, and immunostained for GFP and FLAG to assess Rab41-TOM1L2 binding by Duolink PLA. Representative confocal single-slice images and quantification of the PLA signals. Scale bar, 2 μm. Data are individual values and mean ± SEM ( n = 50 GcAVs examined over three independent experiments). Unpaired two-tailed t test. f Localization of TOM1L2 domain deletion constructs. HeLa cells were transfected with mCherry-LC3 and mClover-TOM1L2 full-length (FL), ΔVHS, or ΔGAT. Shown are representative confocal single-slice images of three independent experiments. Scale bar, 10 μm. Source data are provided as a Source Data file.

Article Snippet: For immunoblot analysis, the following antibodies were used as a primary antibody: streptolysin O (SLO) (ab188539; Abcam, 1:1000), HA (HA124; Nacalai Tesque, 1:1000), FLAG (M2; A2220; Sigma-Aldrich, 1:1000), GFP (GF200; 04363-24; Nacalai Tesque, 1:1000), MBP (E8032; New England BioLabs, 1:1000), GAPDH (sc-47724; Santa cruz biotechnology), Rab8A (D22D8; 6975 S; Cell Signaling Technology, 1:1000), Rab41 (NBP2-83434; NOVUS Biologicals, 1:1000), Rab41 (18818-1-AP; Proteintech Group, 1:500), VPS4A/B (17673-1-AP, Proteintech Group, 1:1000), and TOM1L2 (GTX106295; GENETEX, 1:1000).

Techniques: Expressing, Mass Spectrometry, Two Tailed Test, Infection, Staining, Co-Immunoprecipitation Assay, Incubation, Immunoprecipitation, Western Blot, Binding Assay, Construct, Transfection

Journal: Nature Communications

Article Title: Rab41-mediated ESCRT machinery repairs membrane rupture by a bacterial toxin in xenophagy

doi: 10.1038/s41467-023-42039-2

Figure Lengend Snippet: a , b HeLa cells expressing EmGFP-Rab41 and mCherry-LC3 were transfected with the indicated siRNA, and infected with GAS for 4 h. Cellular and bacterial DNA were stained with DAPI. Representative confocal single-slice images ( a ) and quantification of Rab41 recruitment to GcAVs ( b ). Scale bar, 10 μm. Data in ( b ) are individual values and mean ± SEM ( n = 30 GcAVs examined over three independent experiments). One-way ANOVA, Dunnett’s test. c , d HeLa cells expressing GFP-LC3 were transfected with the indicated siRNA, and infected with GAS for 4 h. Endogenous VPS4 was visualized with an anti-VPS4 antibody, and cellular and bacterial DNA were stained with DAPI. Representative confocal single-slice images ( c ) and quantification of VPS4 recruitment to GcAVs ( d ). Scale bar, 10 μm. Data in ( b ) are individual values and mean ± SEM ( n = 30 GcAVs examined over three independent experiments). One-way ANOVA, Dunnett’s test. e , f HeLa cells expressing GFP-LC3 were transfected with the indicated siRNA, and infected with GAS. Cells were stained with LysoTracker Red 30 min prior to fixation. Representative confocal single-slice images are presented ( e ) and the intensity of LysoTracker within GcAVs was quantified. Scale bar, 10 μm. Data in ( f ) are individual values and mean ± SEM ( n = 30 GcAVs examined over three independent experiments). g Intracellular bacterial CFU at 6 hpi in the control and TOM1L2-knockdown cells. Data are individual values and mean ± SEM ( n = 3 biologically independent experiments). One-way ANOVA, Dunnett’s test. h , i HeLa WT or TOM1L2 knockout cells expressing mCherry-LC3 and EmGFP-Rab41 were infected with GAS for 4 h, fixed, and stained with DAPI. Representative confocal single-slice images ( h ) and quantification of Rab41-positive GcAVs ( i ). Scale bar, 10 μm. Data in ( i ) are individual values and mean ± SEM from three independent experiments, and 200 cells

Article Snippet: For immunoblot analysis, the following antibodies were used as a primary antibody: streptolysin O (SLO) (ab188539; Abcam, 1:1000), HA (HA124; Nacalai Tesque, 1:1000), FLAG (M2; A2220; Sigma-Aldrich, 1:1000), GFP (GF200; 04363-24; Nacalai Tesque, 1:1000), MBP (E8032; New England BioLabs, 1:1000), GAPDH (sc-47724; Santa cruz biotechnology), Rab8A (D22D8; 6975 S; Cell Signaling Technology, 1:1000), Rab41 (NBP2-83434; NOVUS Biologicals, 1:1000), Rab41 (18818-1-AP; Proteintech Group, 1:500), VPS4A/B (17673-1-AP, Proteintech Group, 1:1000), and TOM1L2 (GTX106295; GENETEX, 1:1000).

Techniques: Expressing, Transfection, Infection, Staining, Control, Knockdown, Knock-Out, Two Tailed Test

Journal: Journal of extracellular biology

Article Title: Biogenesis of JC polyomavirus associated extracellular vesicles

doi: 10.1002/jex2.43

Figure Lengend Snippet: Disruption of neutral sphingomyelinase 2 reduces EV mediated spread of JCPyV. (A) SVG-A cells were infected with JCPyV then treated with either DMSO vehicle control or 10 μ M cambinol. Spread of virus was evaluated at 3, 6, and 9 days post infection (dpi) for %VP1(+) cells. (B) shRNA targeting SMPD3 or an empty vector was used to produce control (CTRL) or SMPD3 knockdown (KD) cell lines. RT-qPCR was used to confirm sufficient knockdown of gene expression. (C) CTRL or SMPD3 KD cells were infected with virus and viral spread was evaluated at 3, 6, and 9 dpi. (D) Nanoparticle tracking analysis (NTA) was used to evaluate the concentration of particles/mL and compared to the initial cell count the supernatant was harvested from to determine particles produced per cell. Values are compared to CTRL. (E) Transmission electron microscopy (TEM) was used to evaluate virus(+) EVs derived from CTRL and SMPD3 KD cells. Scale bars are 200 nm. (F) Western blot analysis was used to evaluate relative purity of EV samples. 7.5 μ g of whole cell lysate (WCL), EVs derived from CTRL and SMPD3 KD cells were run and probed for common EV markers (ALIX, Flotillin-1, Annexin V, CD9, LC3A/B-I and -II), potential contaminants (GM130 and ApoA1), and the major viral capsid protein VP1. ApoA1 was not detected in any lane. (G) Virus(+) EVs derived from CTRL or SMPD3 KD lines were used to infect naïve wild-type SVG-A cells and evaluated for infection after 3 days. (H) qPCR was used to evaluate the quantity of viral genomes associated with EVs derived from CTRL or SMPD3 KD cells. (I) EVs were labeled with PKH67 and used in an uptake assay. Internalization of EVs was determined by flow cytometry before and after a trypan blue quench. Percent PKH67(+) cells from each sample type were normalized to the CTRL EVs for presentation. Only post-trypan blue quench values are shown

Article Snippet: Primary antibodies and respective dilutions for Western blots include annexin V (Abcam, Cambridge, UK ab117439, 1:1000), CD9 (Cell Signaling Techologies, Danvers, MA, CST 13174S, 1:1000), CD81 (Systems Biosciences, Palo Alto, CA, 1:1000), Flotillin-1 (CST 18634S, 1:1000), ALIX (CST 3A9, 1:1000) or (CST E6P9B, 1:1000), GRASP65 (Thermo PA3910, 1:5000), RAB8A (CST D22D8, 1:1000), RAB27A (CST D7Z9Q, 1:1000), LC3A/B (CST D3U4C, 1:1000), β -actin (CST 8H10D10, 1:1000), and PAB597 (purified, 1:2000).

Techniques: Disruption, Infection, Control, Virus, shRNA, Plasmid Preparation, Knockdown, Quantitative RT-PCR, Gene Expression, Concentration Assay, Cell Counting, Produced, Transmission Assay, Electron Microscopy, Derivative Assay, Western Blot, Labeling, Flow Cytometry

Journal: Journal of extracellular biology

Article Title: Biogenesis of JC polyomavirus associated extracellular vesicles

doi: 10.1002/jex2.43

Figure Lengend Snippet: CRISPR/Cas9 knockout of SMPD3 decreases JCPyV(+) EV. (A) SVG-A cells were targeted with a CRISPR/Cas9 system to knockout SMPD3. Two clones were grown and sequenced against wild-type SVG-A cells. Sequencing results around the induced mutations are presented against the recorded NCBI sequence for SMPD3. The purple box represents the target sequence carried by the guideRNA. Populations (P#) represent CRISPR variants detected during next generation sequencing, with two major populations detected for KO1 and three for KO2. Graphic created in Unipro UGENE . (B) Wild-type (WT) or knockout (KO) cells were infected with JCPyV and evaluated for initial viral infection at 3 dpi. (C) EVs were evaluated for particles produced per cell by comparing the NTA data to the initial cell count. Values are compared to WT. (D) Negative stains paired with TEM was used to evaluate virus(+) EVs derived from WT, KO1, and KO2 cells. Scale bars are 200 nm. (E) Western blot analysis was used to see presence of EV markers and absence of potential contaminants across all EVs compared to whole cell lysate (WCL). 1.8 μ g of each sample was probed for common EV markers (ALIX, Flotillin-1, Annexin V, CD9), potential contaminants (GM130 and ApoA1), and the major viral capsid protein VP1. ApoA1 was not detected in any lane. (F) Virus(+) EVs derived from each cell line were used to infect naïve wild-type SVG-A cells and evaluated for infection after 3 days. (G) qPCR was used to evaluate the quantity of viral genomes associated with EVs derived from each line. (H) Infectious EVs derived from each cell line were labeled with PKH67 and used in an uptake assay. Internalization of EVs was determined by flow cytometry before and after a trypan blue quench. Percent PKH67(+) cells from each sample type were normalized to the WT EVs for presentation, showing only post-trypan blue quench values. Graph represents two independent experiments in triplicate

Article Snippet: Primary antibodies and respective dilutions for Western blots include annexin V (Abcam, Cambridge, UK ab117439, 1:1000), CD9 (Cell Signaling Techologies, Danvers, MA, CST 13174S, 1:1000), CD81 (Systems Biosciences, Palo Alto, CA, 1:1000), Flotillin-1 (CST 18634S, 1:1000), ALIX (CST 3A9, 1:1000) or (CST E6P9B, 1:1000), GRASP65 (Thermo PA3910, 1:5000), RAB8A (CST D22D8, 1:1000), RAB27A (CST D7Z9Q, 1:1000), LC3A/B (CST D3U4C, 1:1000), β -actin (CST 8H10D10, 1:1000), and PAB597 (purified, 1:2000).

Techniques: CRISPR, Knock-Out, Clone Assay, Sequencing, Next-Generation Sequencing, Infection, Produced, Cell Counting, Virus, Derivative Assay, Western Blot, Labeling, Flow Cytometry

Journal: Journal of extracellular biology

Article Title: Biogenesis of JC polyomavirus associated extracellular vesicles

doi: 10.1002/jex2.43

Figure Lengend Snippet: Knockdown of tetraspanin CD9 or CD81 releases fewer infectious EVs. (A) SVG-A cells were targeted with shRNA against CD9 or CD81. RT-qPCR was used to confirm sufficient knockdown compared to control line. (B) Depletion of respective proteins (left: CD9 KD, right: CD81 KD) was confirmed using Western blot analysis with antibodies against CD9, CD81, and β -actin. (C) Spread of virus was evaluated at 3, 6, and 9 dpi in knockdown versus control cells. (D) EVs harvested from infected cells were assessed via NTA and initial cell counts for particles produced per cell. Each was normalized to control. (E) EV morphology and spatial relationship to JCPyV particles was observed by TEM using a negative stain. Scale bars are 200 nm. (F) EVs were characterized by Western blot analysis and probed for common EV markers (ALIX, Flotillin-1, Annexin V, and CD9), potential contaminants (GM130 and ApoA1), and the viral protein VP1. ApoA1 was not detected in any lane. (G) EVs derived from CTRL or either knockdown line were used in an EV-reinfection assay with infectivity evaluated at 3 dpi for %VP1(+) cells. Values are normalized to control for representation. (H) Viral genomes associated with EVs derived from each line were calculated using qPCR. (I) EVs were labeled with PKH67 and tested for uptake potential by flow cytometry before and after a trypan blue quench. Percent PKH67(+) cells (internalized EVs) post trypan blue quench are shown compared to the control for each sample. Graph represents two independent experiments run in triplicate

Article Snippet: Primary antibodies and respective dilutions for Western blots include annexin V (Abcam, Cambridge, UK ab117439, 1:1000), CD9 (Cell Signaling Techologies, Danvers, MA, CST 13174S, 1:1000), CD81 (Systems Biosciences, Palo Alto, CA, 1:1000), Flotillin-1 (CST 18634S, 1:1000), ALIX (CST 3A9, 1:1000) or (CST E6P9B, 1:1000), GRASP65 (Thermo PA3910, 1:5000), RAB8A (CST D22D8, 1:1000), RAB27A (CST D7Z9Q, 1:1000), LC3A/B (CST D3U4C, 1:1000), β -actin (CST 8H10D10, 1:1000), and PAB597 (purified, 1:2000).

Techniques: Knockdown, shRNA, Quantitative RT-PCR, Control, Western Blot, Virus, Infection, Produced, Staining, Derivative Assay, Labeling, Flow Cytometry

Journal: Journal of extracellular biology

Article Title: Biogenesis of JC polyomavirus associated extracellular vesicles

doi: 10.1002/jex2.43

Figure Lengend Snippet: Single knockdown of ESCRT related proteins does not affect JCPyV(+) EV production. (A) shRNA was used to knockdown seven separate ESCRT-related proteins. Gene knockdown was confirmed by RT-qPCR for the respective gene compared to control cells. (B) Each knockdown line was infected with JCPyV to evaluate spread of virus over 3, 6, and 9 days. (C) Particles produced per cell was determined using NTA for EV particle concentration compared to the initial cell count. Values are normalized to control. (D) TEM was used to examine EV morphology and spatial relationship to JCPyV particles using a negative stain. Scale bar = 200 nm. (E) EVs derived from each cell line were characterized by Western blot analysis and probed for EV markers (ALIX, HSC70, Flotillin-1, Annexin V, CD9, LC3A/B-I and -II), potential EV contaminants (GM130), and the viral protein VP1. ApoA1 was tested on a separate Western and not detected in any lane. (F) Virus(+) EVs derived from each cell line was evaluated for re-infection potential at 3 dpi. Percent VP1(+) cells were each normalized to control. (G) qPCR was used to determine the quantity of viral genomes associated with EVs

Article Snippet: Primary antibodies and respective dilutions for Western blots include annexin V (Abcam, Cambridge, UK ab117439, 1:1000), CD9 (Cell Signaling Techologies, Danvers, MA, CST 13174S, 1:1000), CD81 (Systems Biosciences, Palo Alto, CA, 1:1000), Flotillin-1 (CST 18634S, 1:1000), ALIX (CST 3A9, 1:1000) or (CST E6P9B, 1:1000), GRASP65 (Thermo PA3910, 1:5000), RAB8A (CST D22D8, 1:1000), RAB27A (CST D7Z9Q, 1:1000), LC3A/B (CST D3U4C, 1:1000), β -actin (CST 8H10D10, 1:1000), and PAB597 (purified, 1:2000).

Techniques: Knockdown, shRNA, Quantitative RT-PCR, Control, Infection, Virus, Produced, Concentration Assay, Cell Counting, Staining, Derivative Assay, Western Blot

Journal: Journal of extracellular biology

Article Title: Biogenesis of JC polyomavirus associated extracellular vesicles

doi: 10.1002/jex2.43

Figure Lengend Snippet: Secretory autophagy related proteins contribute to infectious JCPyV(+) EV populations. (A) RT-qPCR was used to confirm sufficient knockdown of RAB8A, RAB27A, or GRASP65 compared to control cells. (B) Depletion of respective proteins was confirmed using Western blot analysis using antibodies against RAB8A, RAB27A, GRASP65, and β -actin. Top panel shows CTRL vs RAB8A KD, middle panel shows CTRL vs RAB27A KD, bottom panel shows CTRL vs GRASP65 KD. (C) Spread of JCPyV was evaluated at 3, 6, and 9 dpi in KD versus CTRL cells. (D) Particles produced per cell for each EV population was assessed using NTA against the initial cell count. Each is normalized to control. (E) EV morphology and spatial relationship to JCPyV particles was observed by TEM. Scale bar = 200 nm. (F) EV characterization via Western blot analysis with common EV markers (ALIX, Flotillin-1, Annexin V, and CD9), potential contaminants (GM130 and ApoA1), and the viral protein VP1 was performed for each EV against WCL. ApoA1 was not detected in any lane. (G) EVs were used to infect naïve SVG-A cells with infectivity evaluated at 3 dpi for %VP1(+) cells. Values are normalized to control for representation. (H) Viral genomes associated with EVs derived from each line were calculated using absolute qPCR. (I) EVs were labeled with PKH67 and tested for uptake potential by flow cytometry before and after a trypan blue quench. Percent PKH67(+) cells (internalized EVs) post trypan blue quench are shown compared to the control EVs for each sample

Article Snippet: Primary antibodies and respective dilutions for Western blots include annexin V (Abcam, Cambridge, UK ab117439, 1:1000), CD9 (Cell Signaling Techologies, Danvers, MA, CST 13174S, 1:1000), CD81 (Systems Biosciences, Palo Alto, CA, 1:1000), Flotillin-1 (CST 18634S, 1:1000), ALIX (CST 3A9, 1:1000) or (CST E6P9B, 1:1000), GRASP65 (Thermo PA3910, 1:5000), RAB8A (CST D22D8, 1:1000), RAB27A (CST D7Z9Q, 1:1000), LC3A/B (CST D3U4C, 1:1000), β -actin (CST 8H10D10, 1:1000), and PAB597 (purified, 1:2000).

Techniques: Quantitative RT-PCR, Knockdown, Control, Western Blot, Produced, Cell Counting, Infection, Derivative Assay, Labeling, Flow Cytometry

Journal: Journal of extracellular biology

Article Title: Biogenesis of JC polyomavirus associated extracellular vesicles

doi: 10.1002/jex2.43

Figure Lengend Snippet:

Article Snippet: Primary antibodies and respective dilutions for Western blots include annexin V (Abcam, Cambridge, UK ab117439, 1:1000), CD9 (Cell Signaling Techologies, Danvers, MA, CST 13174S, 1:1000), CD81 (Systems Biosciences, Palo Alto, CA, 1:1000), Flotillin-1 (CST 18634S, 1:1000), ALIX (CST 3A9, 1:1000) or (CST E6P9B, 1:1000), GRASP65 (Thermo PA3910, 1:5000), RAB8A (CST D22D8, 1:1000), RAB27A (CST D7Z9Q, 1:1000), LC3A/B (CST D3U4C, 1:1000), β -actin (CST 8H10D10, 1:1000), and PAB597 (purified, 1:2000).

Techniques: Sequencing

Journal: Journal of extracellular biology

Article Title: Biogenesis of JC polyomavirus associated extracellular vesicles

doi: 10.1002/jex2.43

Figure Lengend Snippet:

Article Snippet: Primary antibodies and respective dilutions for Western blots include annexin V (Abcam, Cambridge, UK ab117439, 1:1000), CD9 (Cell Signaling Techologies, Danvers, MA, CST 13174S, 1:1000), CD81 (Systems Biosciences, Palo Alto, CA, 1:1000), Flotillin-1 (CST 18634S, 1:1000), ALIX (CST 3A9, 1:1000) or (CST E6P9B, 1:1000), GRASP65 (Thermo PA3910, 1:5000), RAB8A (CST D22D8, 1:1000), RAB27A (CST D7Z9Q, 1:1000), LC3A/B (CST D3U4C, 1:1000), β -actin (CST 8H10D10, 1:1000), and PAB597 (purified, 1:2000).

Techniques: