Journal: bioRxiv

Article Title: Active EB1 surges promote tubulin influx into the growing outer segments of the bipartite olfactory cilia in Drosophila

doi: 10.1101/2024.09.10.612170

Figure Lengend Snippet: A) Copurification of EB1:GFP from the chaGal4>EB1:GFP expressing Drosophila head extracts with the recombinant Glutathione S-transferase (GST)-tagged, KLP68D tail (GST-KLP68DT) fragment using affinity chromatography. The arrow indicates the EB1:GFP band. B) The immune-coprecipitation (IP) of the recombinant KLP68D from the head extracts of chaGal4>Klp68D:YFP and chaGal4>Klp68D(ΔT)YFP, expressing UAS-EB1FLAG using anti-FLAG. The arrow indicates a full-length KLP68DYFP band.

Article Snippet: Proteins from these gels were transferred onto a previously activated PVDF membrane (Hybond-P, GE Healthcare Ltd) in an electro-blotting apparatus (Bio-Rad, USA) following the supplier’s protocol and incubated in different primary antisera solutions as the following: anti-GFP Rabbit (dilution, 1:500; #3999 Bio Vision Inc., CA, USA) or anti-FLAG Rabbit (1:500; #F7425 Sigma-Aldrich, USA) Or anti-GST mouse (1:1000, Bioklone Biotech Pvt.

Techniques: Copurification, Expressing, Recombinant, Affinity Chromatography

Journal: bioRxiv

Article Title: Actomyosin contractility modulates Wnt signaling through adherens junction stability

doi: 10.1101/220178

Figure Lengend Snippet: (A) Salivary glands from control or dpp>flw-RNAi stained for fz3 expression. (B) Localization of Dsh-GFP and FLAG-Axin in the proximal cells of the salivary gland, identified in the dashed line area of (A), (C-C”) Effects of flw-RNAi on ectopic Dll-lacZ in wing imaginal discs: (C) RFP marked flip-out clones expressing Fz-Arr and GFP or flw-RNAi (C) GFP-positive axin nul1 MARCM clones and flw-RNAi in axin nul1 MARCM clones. (C”) Arm S10 flip out clones with GFP or flw-RNAi. (D,D’) Effects of flw-RNAi on Arm distribution in GFP-marked axin null cells. (D) DAPI was used to identify nuclei, and F-actin to mark the edges of the cell. (D’) Percent of nuclear Arm in cells was measured as an intensity plot (dotted line D) in wild type (n = 16), axin nul1 (n = 20), and axin null , flw-RNAi cells (n = 15). Data presented as mean ± SEM; ***p< 0.001. Scale bars: (A) 100 μm, (B-C”) 50 μm, (D) 5 μm.

Article Snippet: The following primary antibodies and dilutions were used: mouse anti-β-galactosidase (1:2000 Promega), mouse anti-Wg (1:100 DSHB), mouse anti-Arm (1:50 DSHB), rabbit anti-cleaved Caspase 3 (1:100 Cell Signaling), guinea pig anti-Sens (1:500, a gift from Hugo Bellen, Dept. of Molecular and Human Genetics, Baylor College of Medicine, USA), mouse anti-Dll (1:300, a gift from Ian Duncan, Dept. of Biology, Washington University in St. Louis, USA), rabbit anti-Phospho-Myosin Light Chain 2 (Ser19) (p-MyoII) (1:25 Cell Signaling), mouse anti-GFP (1:500, Cell Signaling), rabbit anti-FLAG (1:200, Sigma), rat anti-DEcad (extracellular domain) (1:50 DSHB), rat anti-Ci (1:50, DSHB).

Techniques: Staining, Expressing, Clone Assay

Journal: bioRxiv

Article Title: Actomyosin contractility modulates Wnt signaling through adherens junction stability

doi: 10.1101/220178

Figure Lengend Snippet: (A,B) Wnt-responsive luciferase TOPFLASH assay measuring TCF/LEF reporter activity in (A) MCF7 and (B) RKO cells following Wnt3A transfection and siPP1β and siMYPT3 transfection. Data are presented as mean ± SEM with letters above representing significant difference from corresponding column, (P<0.01). (C) Western blot analysis of total cellular levels of E-cad, β-cat, MYPT3, Wnt3A and PP1β. β-tub was used as a loading control. (D) Western blot analysis of β-cat in cytoplasmic (Cyto), membranous (Mem), chromatin-bound (CB), and whole cell extract (WCE) fractions in MCF7 cells. GAPDH, Na + /K + ATPase, and Histone 3 were used as loading controls for corresponding fractions. Complete fraction, see . (E) Quantification of relative levels of β-cat from each fraction taken from (D), normalized to Control + siScramble conditions. Data shown as mean ± SEM (n = 4 biological replicates), ns = not significant, *P<0.05.

Article Snippet: Proteins were transferred onto nitrocellulose membranes, and probed against the following primary antibodies: rabbit anti-β-catenin (1:1000, Cell Signaling), rabbit anti-E-cadherin (1:1000, Cell Signaling), mouse anti-PPP1R16A (MYPT-3) (1:500, abcam), mouse anti-Protein Phosphatase 1 beta (PP1β) (1:1000, abcam), rabbit anti-Wnt3A (1:1000, Cell Signaling), mouse anti-β-tubulin (1:1000, ABM), rabbit anti-GAPDH (1:3000, Cell Signaling), mouse anti-Na+/K+ ATPase (1:50 DSHB), rabbit anti-Histone H3 (1:1000 Cell Signaling).

Techniques: Luciferase, TOPFlash assay, Activity Assay, Transfection, Western Blot, Control

Journal: bioRxiv

Article Title: Genome-wide analysis of consistently RNA edited sites in human blood reveals interactions with mRNA processing genes and suggests correlations with cell types and biological variables

doi: 10.1101/254045

Figure Lengend Snippet: Association between gene expression and CES total editing rate We analyzed association between CES total editing rate and gene expression for 14,961 human genes. (a) Gene set enrichment analysis by hypergeometric test on GO-BP categories and REACTOME pathways revealed that associated genes are mainly involved in immune system response mediated by interferon I and alpha / beta. (b) When we analyze distribution of CES total editing rate and ADAR gene expression, ADAR expression levels explains ∼ 13% of observed variability. No significant effect is observed for ADARB1 expression. ADAR and ADARB1 expression levels are reported as residuals of ridge regression with technical covariates (see description of data in methods section). The graphs report adjusted p-value and R2 value from robust regression analysis. (c) The 1,122 genes associated to CES total editing rate after removing ADAR expression effect were enriched for genes mainly involved in ribonucleoprotein and RNA processing.

Article Snippet: During the immunoblot step the following primary antibodies were used 1h at RT: mouse anti-ADAR1 (Santa Cruz, cod. sc-73408) 1:300 in 5% non-fat dry milk in TBST 0,1%; mouse anti-IFI16 (Abcam, cod.

Techniques: Expressing

Journal: bioRxiv

Article Title: Genome-wide analysis of consistently RNA edited sites in human blood reveals interactions with mRNA processing genes and suggests correlations with cell types and biological variables

doi: 10.1101/254045

Figure Lengend Snippet: Genes associated with CES total editing rate are enriched for ADAR interactors (a) Reconstructed PPI network including ADARs and proteins encoded by best genes significantly associated with global editing levels (FDR < 0.01). Among these proteins, we observed 285 potential ADARs interactors, including 9 direct partners of ADARs proteins. (b) Boxplot of number of ADARs interacting genes observed in 1M random simulations. The observed number of interactions (285) resulted in empirical p-value < 1e-6. (c) ADARs interactors are strongly enriched for RNA binding proteins in GO-MF categories. (d) Distribution of degree and betweenness centrality values among network nodes are represented by violin plots. ADAR1 protein has a major role (higher values) among ADAR proteins. Among ADARs direct partners, ELAVL1, RPA1 and IFI16 showed high values of degree and betweenness centrality, suggesting a central role in the network. (e) ADAR1 interaction with RPA70 (coded by RPA1) and IFI16 determined by co-immunoprecipitation. After immunoprecipitation with ADAR1 antibody, western blot for IFI16 and RPA70 are reported. For a better discrimination two times of exposure are reported in the figure.

Article Snippet: During the immunoblot step the following primary antibodies were used 1h at RT: mouse anti-ADAR1 (Santa Cruz, cod. sc-73408) 1:300 in 5% non-fat dry milk in TBST 0,1%; mouse anti-IFI16 (Abcam, cod.

Techniques: RNA Binding Assay, Immunoprecipitation, Western Blot

Journal: bioRxiv

Article Title: Genome-wide analysis of consistently RNA edited sites in human blood reveals interactions with mRNA processing genes and suggests correlations with cell types and biological variables

doi: 10.1101/254045

Figure Lengend Snippet: Impact of cell composition on CES total editing rate and ADAR / ADARB1 expression Our analysis revealed strong associations with CES total editing rate for 4 cell type variables (a), representing proportion of neutrophils, monocytes, dendritic cells (DC) and T helper (Th). Specific cell variables resulted significantly associated also to ADAR (b) and ADARB1 (c) expression. Significance level (p) and correlation coefficient (r) are reported in each plot based on Pearson’s product-moment. Only non-zero observations are plotted.

Article Snippet: During the immunoblot step the following primary antibodies were used 1h at RT: mouse anti-ADAR1 (Santa Cruz, cod. sc-73408) 1:300 in 5% non-fat dry milk in TBST 0,1%; mouse anti-IFI16 (Abcam, cod.

Techniques: Expressing

Journal: bioRxiv

Article Title: Genome-wide analysis of consistently RNA edited sites in human blood reveals interactions with mRNA processing genes and suggests correlations with cell types and biological variables

doi: 10.1101/254045

Figure Lengend Snippet: Impact of biological / pharmacological factors on CES total editing rate and ADAR / ADARB1 expression Our analysis revealed significant associations with CES total editing rate for blood pressure medication, BMI current, Age and Sex (a). Specific biological variables resulted significantly associated also to ADAR (b) and ADARB1 (c) expression. Significance level of association after correction for cell composition is reported (p (cell)) is reported in each plot based on Mann-Whitney-Wilcoxon or Pearson’s product-moment correlation test for binary and continuous variables, respectively. For continuous variables the Pearson correlation coefficient (r) is also reported.

Article Snippet: During the immunoblot step the following primary antibodies were used 1h at RT: mouse anti-ADAR1 (Santa Cruz, cod. sc-73408) 1:300 in 5% non-fat dry milk in TBST 0,1%; mouse anti-IFI16 (Abcam, cod.

Techniques: Expressing, MANN-WHITNEY

Journal: bioRxiv

Article Title: Genome-wide analysis of consistently RNA edited sites in human blood reveals interactions with mRNA processing genes and suggests correlations with cell types and biological variables

doi: 10.1101/254045

Figure Lengend Snippet: Impact of cell composition, biological and pharmacological factors on PCs of editing levels The heathmap represents strength of association between the first 5 principal components of CESs (PCs) and ADAR / ADARB1 expression (upper panel), 7 cell composition variables (middle panel) and 11 biological / pharmacological variables (lower panel). Only factors showing significant association with at least one of the first 5 PCs are represented. Significant p values (< 0.05) are colored in yellow-red scale, while p value > 0.05 are represented in grey scale. Age, BMI, blood pressure medications, smoke and alcohol all associate with PC1. Also time of blood draw seems to have a small, but consistent effect, on different PCs. For each PC, variance explained is represented by the bar plot in the upper side.

Article Snippet: During the immunoblot step the following primary antibodies were used 1h at RT: mouse anti-ADAR1 (Santa Cruz, cod. sc-73408) 1:300 in 5% non-fat dry milk in TBST 0,1%; mouse anti-IFI16 (Abcam, cod.

Techniques: Expressing

Journal: bioRxiv

Article Title: Genome-wide analysis of consistently RNA edited sites in human blood reveals interactions with mRNA processing genes and suggests correlations with cell types and biological variables

doi: 10.1101/254045

Figure Lengend Snippet: Association study for SNPs and CES total editing rate (a) Manhattan plot representing the association between 573,801 SNPs and CES total editing rate, where black line represents threshold for the top 100 SNPs (p value ∼ 10e-4). (b) Detailed view of genotyped SNPs located in the region at chromosome 7 that showed significant association with CES total editing rate. Known GWAS associations for human phenotypes from GRASP database are reported in the lower panel. (c) The top associated SNP (rs856554) showed a significant effect on global editing level, while no significant correlation was observed with ADAR and ADARB1 expression. (d) Real-time expression analysis of ADAR and ADARB1 mRNA after B-EBV transfection of LOC730338. Not transfected cells were used as control samples. Data are reported as 2 -ΔΔ ct (expression level of control sample is equal to 1) and represent mean values and standard errors obtained from at least 3 independent evaluations. Unpaired t test was used for statistical analysis (*p< 0.05).

Article Snippet: During the immunoblot step the following primary antibodies were used 1h at RT: mouse anti-ADAR1 (Santa Cruz, cod. sc-73408) 1:300 in 5% non-fat dry milk in TBST 0,1%; mouse anti-IFI16 (Abcam, cod.

Techniques: Expressing, Transfection