human rab25  (Bio-Rad)

Journal: Communications Biology

Article Title: Analysis of the glyco-code in pancreatic ductal adenocarcinoma identifies glycan-mediated immune regulatory circuits

doi: 10.1038/s42003-021-02934-0

Figure Lengend Snippet: List of Antibodies used in this paper.

Article Snippet: Purified anti-human RAB25 , Cell Signaling , 13048 T , 1:500.

Techniques: Flow Cytometry, Microscopy, Western Blot, Purification, Enzyme-linked Immunosorbent Assay

Journal: Angiogenesis

Article Title: Notch regulates vascular collagen IV basement membrane through modulation of lysyl hydroxylase 3 trafficking

doi: 10.1007/s10456-021-09791-9

Figure Lengend Snippet: Rab10 and Rab25 work in combination to traffic LH3 to CIVC vesicles. ( a ) Representative images of ECs expressing RFP-Rab10 wild-type (WT) and stained for collagen IV (Col IV) (green). Green arrowheads indicate collagen IV-containing (CIVC) vesicles only and yellow arrowheads indicate Rab10 puncta only. ( b ) Representative images of scramble, Rab10, Rab25, or LH3 siRNA-treated ECs and stained for Col IV (red), actin (green), and DNA (blue). Arrowheads denote extracellular Col IV secretion. ( c ) Graph of extracellular Col IV ratio of scramble, Rab10, Rab25, or LH3 siRNA-treated ECs cultured in serum-starve (SS) media. ( d ) Representative images of fibrin-bead sprouts between indicated siRNA treatment groups. Sprouts were stained for actin (grey) to delineate sprout morphology. ( e – g ) Graphs of sprouting parameters for scramble, Rab10, Rab25, or LH3 siRNA-treated sprouts. ( h ) Representative images of ECs cultured in VEGF-containing or SS media and stained for Col IV (green), LH3 (red), and DNA (blue). Green arrowheads indicate CIVC vesicles only and yellow arrowheads indicate co-localized puncta. ( i ) Graph of percent CIVC vesicles co-localized with LH3 in ECs cultured in VEGF-containing or SS media. ( j ) Representative images of scramble, Rab10, Rab25, or both Rab10/25 siRNA-treated ECs and stained for Col IV (green), LH3 (red), actin (grey), and DNA (blue). Yellow arrowheads indicate co-localized puncta only, green arrowheads indicate Col IV only puncta, and red arrowheads indicate LH3 puncta only. ( k ) Graph of percent CIVC vesicles co-localized with LH3 in scramble, Rab10, Rab25, or LH3 siRNA-treated conditions cultured in SS media or SS media with CLQ (10 µM). ( l ) Representative images of ECs expressing RFP-Rab10 WT, RFP-Rab10 constitutively active (CA), or RFP-Rab10 dominant negative (DN), stained for Col IV (green) and LH3 (blue). Yellow arrowheads indicate co-localized puncta only, green arrowheads indicate Col IV puncta, and red arrowheads indicate LH3 puncta. ( m ) Graph of percent CIVC vesicles co-localized with LH3 in ECs expressing RFP-Rab10 WT/CA/DN. ( n ) Graph of percent Rab10 wild-type (WT) puncta co-localized with Rab25 in either VEGF-containing or SS media. ( o ) Graph of percent Rab10 puncta co-localized with Rab25 in ECs transfected with indicated constructs. ( p ) Schematic diagram showing how Rab10 and Rab25 function to coordinate delivery of LH3 to CIVC vesicles for proper secretion of Col IV. For all graphs n number of cells unless otherwise indicated. For all experiments, data represented as mean ± 95% confidence intervals. Black bars indicate comparison groups with indicated p -values. All p -values are from two-tailed Student’s t -test from at least three experiments. * p ≤ 0.05; ** p ≤ 0.01; *** p ≤ 0.001; **** p ≤ 0.0001; ns not significant

Article Snippet: Full-length human Rab25 cDNA was purchased from Origene (RC203413, ORIgene) and cloned into pME-MCS as described supplementary table 1.

Techniques: Expressing, Staining, Cell Culture, Dominant Negative Mutation, Transfection, Construct, Two Tailed Test

Journal: Angiogenesis

Article Title: Notch regulates vascular collagen IV basement membrane through modulation of lysyl hydroxylase 3 trafficking

doi: 10.1007/s10456-021-09791-9

Figure Lengend Snippet: Notch1 deficient mouse retinas have reduced collagen IV. ( a ) Representative images of wild-type (WT; Cdh5-PAC-CreER +/− ; Notch +/+ ), Notch EC knockout (ecko)/− (HET, Cdh5-PAC-CreER +/− ; Notch flox/+ ), or Notch ecko/ecko (NULL, Cdh5-PAC-CreER +/− ; Notch flox/flox ) retinas harvested at P5 and stained for isolectin B4 (IB4). ( b ) Graph of vasculature area for vessels between indicate genotypes. ( c ) Graph of number of branch points at vascular front. ( d ) Top- schematic of Col IV measurements taken on sprouts. Graph of collagen IV (Col IV) intensity starting at the vascular front and measured back every 50 μm between indicated groups. Intensities are normalized to Col IV levels at 200 μm for each group. ( e ) Representative images of retinas harvested at P5 and stained for Col IV (red), DNA (blue), and IB4 (green) to identify blood vessels between indicated genotypes. ( f ) Graph of Col IV fluorescence intensity across indicated groups. ( g ) Model- VEGF-binding at the tip cells decreases Notch activation. In stalk cells, Notch activation promotes expression of DENNd4c which activates Rab10. Active Rab10 works with Rab25 to traffic Lysyl hydroxylase 3 (LH3) to Col IV-containing vesicles, allowing for secretion of Col IV into the extracellular space. For all experiments, data represented as mean ± 95% confidence intervals. N number of measurements. In all conditions, 6 or more mice were used per group. Black bars indicate comparison groups with indicated p -values. All p -values are from two-tailed Student’s t-test from duplicate experiments. * p ≤ 0.05; ** p ≤ 0.01; *** p ≤ 0.001; **** p ≤ 0.0001; ns, not significant

Article Snippet: Full-length human Rab25 cDNA was purchased from Origene (RC203413, ORIgene) and cloned into pME-MCS as described supplementary table 1.

Techniques: Knock-Out, Staining, Fluorescence, Binding Assay, Activation Assay, Expressing, Two Tailed Test

Journal: Nature

Article Title: In Vivo Base Editing Rescues Hutchinson-Gilford Progeria Syndrome in Mice

doi: 10.1038/s41586-020-03086-7

Figure Lengend Snippet: ( a ) Distribution of all possible single-nucleotide variant (SNV) types in non-tumor liver tissue and liver tumor tissue samples isolated from ABE AAV9-injected and saline-injected mice. Values from individual tissue samples are shown on the left. Aggregated values from all AAV-injected mouse tumor tissue samples, all AAV-injected mouse liver tissue samples, and all saline-injected mouse liver tissue samples are shown on the right, where values represent the mean of each sample type and error bars reflect the standard deviation with each tissue section treated as a different sample: AAV-injected tumor tissue (n=7), AAV-injected liver tissue (n=6), and saline-injected liver tissue (n=2). ( b ) Genomic classification of A•T-to-G•C SNVs. Values from individual tissue samples are shown on the left. Aggregated values from AAV-injected mouse tumor tissue samples, AAV-injected mouse liver tissue samples, and saline-injected mouse liver tissue samples from all tissue types are shown on the right, where values represent the mean of each sample type and error bars reflect the standard deviation with each tissue section treated as a different sample: AAV-injected tumor tissue (n=7), AAV-injected liver tissue (n=6), saline-injected liver tissue (n=2). ( c ) A•T-to-G•C SNVs and indels found in or near genes that are recurrently mutated in human liver cancers , including introns, exons, and at ATAC-seq-defined cis-regulatory regions within 100-kb of each gene’s transcription start site, in AAV-injected mouse tumor tissue samples, AAV-injected mouse liver tissue samples, and saline-injected mouse liver tissue samples. Values represent the mean of individual tissue samples and error bars represent standard deviation. Individual data points are shown for each sample. The complete list of SNVs from ANNOVAR analysis is provided as . Summary statistics for SNV calls are in . ( d ) RNA isolated from mouse liver tissue samples was reverse transcribed and amplified with primer sets specific to mouse Gapdh (detected with Cy5), mouse Actb (detected with Cy5.5), and human RAB25 (detected with TEX 615). Ct values were determined by quantitative PCR and are shown below each lane. N.D. = not detected.

Article Snippet: PCR was carried out using gene-specific primers for mouse Gapdh (Biorad; 10031231), mouse Actb (Biorad; 10031237), and human RAB25 (Biorad; 10031234) with the iQ multiplex powermix (Biorad).

Techniques: Variant Assay, Isolation, Injection, Standard Deviation, Amplification, Real-time Polymerase Chain Reaction

Journal: Virulence

Article Title: MiR-140 inhibits classical swine fever virus replication by targeting Rab25 in swine umbilical vein endothelial cells

doi: 10.1080/21505594.2020.1735051

Figure Lengend Snippet: Sequences of primers, miRNAs, and siRNAs used in this study.

Article Snippet: The membrane was blocked with the polyclonal anti-Rab25 antibody (CSB-PA019175LA01HU, cusabio), and monoclonal anti-β-actin antibody (AB0035, Abways).

Techniques: Sequencing, Over Expression, Knockdown

Journal: Epigenetics

Article Title: RAB25 expression is epigenetically downregulated in oral and oropharyngeal squamous cell carcinoma with lymph node metastasis

doi: 10.1080/15592294.2016.1205176

Figure Lengend Snippet: Strategy to identify epigenetically downregulated genes in pN+ OSCC. On the left: published gene signatures predictive of pN-status in OSCC were used to identified significantly downregulated genes in pN+ OSCC. On the right: MethylCap-Seq was performed on 6 pN0 OSCC and pN+ OSCC. All reads of MCs in gene promoter regions were ranked according to the likelihood of differential methylation and an approximate FDR. The 5,000 MCs with the lowest FDR were further tested by Mann-Whitney-U. The MC associated with genes without annotated gene functions were excluded. In the middle: the gene signature and methylation data were compared to select epigenetically regulated genes in pN+ OSCC (n = 23). From these 23 genes, epigenetically downregulated genes in pN+ OSCC were selected. Based on the amount of mRNA downregulation, statistical differences in methylation between pN0 and pN+ OSCC, and positive and negative predictive value, RAB25 was selected as the most significantly epigenetically downregulated gene in pN+ OSCC compared to pN0 OSCC.

Article Snippet: Antigen retrieval was performed using a citrate buffer (10 mM citric acid, 0.05% Tween 20, pH 6.0) and heated in a microwave oven for 15 min at 300 W. Endogenous peroxidase was blocked with a 0.3% H 2 O 2 solution for 30 min at room temperature, followed by incubation with a mouse monoclonal antibody to human RAB25 clone 3F12F3 (Santa Cruz), diluted 1:50 in PBS with 1% bovine serum albumin, overnight at 4°C.

Techniques: Methylation, MANN-WHITNEY

Journal: Epigenetics

Article Title: RAB25 expression is epigenetically downregulated in oral and oropharyngeal squamous cell carcinoma with lymph node metastasis

doi: 10.1080/15592294.2016.1205176

Figure Lengend Snippet: Epigenetically downregulated genes in pN+ OSCC. All 14 potentially epigenetically downregulated genes in pN+ OSCC compared to pN0 OSCC after cross-reference of expression microarray and MethylCap-Seq data (see Fig. 1 ). The positive and negative predictive value of the reads for pN+ status, associated hypermethylation, read distribution between pN0 and pN+ OSCC, and predictive value of the methylation data are illustrated. P -value for the differential DNA methylation was calculated using the Mann-Whitney-U test. Positive and negative predictive value for the methylation status of all MCs were calculated as follows: OOSCC with a read count of ≥ 3 reads were considered true positives and OOSCC with a count read <3 were considered true negatives. Subsequently, the positive predictive value was then calculated as: (true positive pN+ OOSCC) / (true positive pN+ OOSCC + false positive pN0 OOSCC). Finally, the negative predictive value was calculated as: (true negative pN0 OOSCC) / (true negative pN0 OOSCC + false negative pN+ OOSCC).

Article Snippet: Antigen retrieval was performed using a citrate buffer (10 mM citric acid, 0.05% Tween 20, pH 6.0) and heated in a microwave oven for 15 min at 300 W. Endogenous peroxidase was blocked with a 0.3% H 2 O 2 solution for 30 min at room temperature, followed by incubation with a mouse monoclonal antibody to human RAB25 clone 3F12F3 (Santa Cruz), diluted 1:50 in PBS with 1% bovine serum albumin, overnight at 4°C.

Techniques: Expressing, Microarray, Methylation, DNA Methylation Assay, Mann-Whitney U-Test

Journal: Epigenetics

Article Title: RAB25 expression is epigenetically downregulated in oral and oropharyngeal squamous cell carcinoma with lymph node metastasis

doi: 10.1080/15592294.2016.1205176

Figure Lengend Snippet: RAB25 mRNA levels in relation with the 3 RAB25 TSS 450K probes (cg09243900, cg15896939, and cg19580810) methylation levels in the TCGA OSCC cohort. (A) RAB25 methylation levels compared between OSCC with high RAB25 mRNA levels and OSCC with low RAB25 mRNA levels. The M-values of the 3 RAB25 Infinium 450K promoter probes were significantly higher in OSCC with low RAB25 mRNA z-scores compared to OSCC with high RAB25 mRNA z-scores. (B) Spearman correlations between RAB25 methylation and RAB25 mRNA levels. All 3 RAB25 promoter probes showed a significant negative correlation between RAB25 promoter probe M-values and RAB25 mRNA z-scores.

Article Snippet: Antigen retrieval was performed using a citrate buffer (10 mM citric acid, 0.05% Tween 20, pH 6.0) and heated in a microwave oven for 15 min at 300 W. Endogenous peroxidase was blocked with a 0.3% H 2 O 2 solution for 30 min at room temperature, followed by incubation with a mouse monoclonal antibody to human RAB25 clone 3F12F3 (Santa Cruz), diluted 1:50 in PBS with 1% bovine serum albumin, overnight at 4°C.

Techniques: Methylation

Journal: Epigenetics

Article Title: RAB25 expression is epigenetically downregulated in oral and oropharyngeal squamous cell carcinoma with lymph node metastasis

doi: 10.1080/15592294.2016.1205176

Figure Lengend Snippet: RAB25 expression levels between pN0 and pN+ OSCC in the UMCG and TCGA OSCC cohort. (A) pN+ OSCC in the TCGA cohort (n = 86) express significantly less RAB25 mRNA than pN0 OSCC (n = 61), as revealed by Mann-Whitney-U test. (B) pN + OSCC in the UMCG cohort (n = 87) have significantly less RAB25-positive tumor cells than pN0 OSCC (n = 91), as revealed by Mann-Whitney-U test.

Article Snippet: Antigen retrieval was performed using a citrate buffer (10 mM citric acid, 0.05% Tween 20, pH 6.0) and heated in a microwave oven for 15 min at 300 W. Endogenous peroxidase was blocked with a 0.3% H 2 O 2 solution for 30 min at room temperature, followed by incubation with a mouse monoclonal antibody to human RAB25 clone 3F12F3 (Santa Cruz), diluted 1:50 in PBS with 1% bovine serum albumin, overnight at 4°C.

Techniques: Expressing, MANN-WHITNEY

Journal: Epigenetics

Article Title: RAB25 expression is epigenetically downregulated in oral and oropharyngeal squamous cell carcinoma with lymph node metastasis

doi: 10.1080/15592294.2016.1205176

Figure Lengend Snippet: Correlations between RAB25 expression and tumor characteristics. A) Associations between RAB25 mRNA expression and the clinical characteristics of the TCGA OSCC cohort. B) Associations between RAB25 protein expression and the clinical characteristics of the UMCG OSCC cohort.

Article Snippet: Antigen retrieval was performed using a citrate buffer (10 mM citric acid, 0.05% Tween 20, pH 6.0) and heated in a microwave oven for 15 min at 300 W. Endogenous peroxidase was blocked with a 0.3% H 2 O 2 solution for 30 min at room temperature, followed by incubation with a mouse monoclonal antibody to human RAB25 clone 3F12F3 (Santa Cruz), diluted 1:50 in PBS with 1% bovine serum albumin, overnight at 4°C.

Techniques: Expressing, Biomarker Discovery

Journal: Epigenetics

Article Title: RAB25 expression is epigenetically downregulated in oral and oropharyngeal squamous cell carcinoma with lymph node metastasis

doi: 10.1080/15592294.2016.1205176

Figure Lengend Snippet: Representative examples of RAB25 expression in 2 OSCC as detected by immunohistochemistry. Tissues were scored by the amount of RAB25-positive cells. (A) Example of a well-differentiated OSCC with a high amount of RAB25-expressing cells. (B) Example of a poorly differentiated OSCC with a very low amount of RAB25-positive cells.

Article Snippet: Antigen retrieval was performed using a citrate buffer (10 mM citric acid, 0.05% Tween 20, pH 6.0) and heated in a microwave oven for 15 min at 300 W. Endogenous peroxidase was blocked with a 0.3% H 2 O 2 solution for 30 min at room temperature, followed by incubation with a mouse monoclonal antibody to human RAB25 clone 3F12F3 (Santa Cruz), diluted 1:50 in PBS with 1% bovine serum albumin, overnight at 4°C.

Techniques: Expressing, Immunohistochemistry