Journal: Nucleic Acids Research

Article Title: An internal deletion of ADAR rescued by MAVS deficiency leads to a minute phenotype

doi: 10.1093/nar/gkaa025

Figure Lengend Snippet: Rescue of Adar Δ7-9 by Mavs −/− , Ifih1 −/− and Mavs −/− ; Adarb1 −/− ; Gria2 R/R . ( A) Post-natal survival curves of Adar Δ7-9 ; Mavs −/− , Adar1 Δ7-9 ; Ifih1 −/− and Adar Δ7-9 ; Mavs −/− ; Adarb1 −/− ; Gria2 R/R mice. Expression levels of 5 Interferon stimulated genes measured by qPCR in three different tissues of ( B ) Adar Δ7-9 ; Mavs −/− ( C ) Adar Δ7-9 ; Ifih1 −/− and ( D ) Adar Δ7-9 ; Mavs −/− ; Adarb1 −/− ; Gria2 R/R mice.

Article Snippet: This was done using an antibody against the p150 version of ADAR1 (Synaptic Systems) (Figure ) or by using a pan-ADAR1 antibody (15.8.6 / Santa Cruz) (Figure ).

Techniques: Expressing

Journal: Nucleic Acids Research

Article Title: An internal deletion of ADAR rescued by MAVS deficiency leads to a minute phenotype

doi: 10.1093/nar/gkaa025

Figure Lengend Snippet: ADAR1 variants without exons 7–9 can be expressed in cells. ( A ) Illustration of the p150 and p110 isoforms of ADAR1 indicating the deleted parts of the Adar Δ7-9 allele (red box). The truncation affects only the third double-stranded RNA binding domain (dsRBD), the embedded nuclear localization signal (NLS) and the deaminase domain. Z-DNA binding domains (Zα and Zβ), the nuclear export signal (NES), dsRBD 1 and 2 as well as a C-terminal NLS motif are unaffected. Amplicons tested for detecting truncation are indicated. ( B ) RT-PCR analysis of wildtype and Adar Δ7-9 transcript in primary MEFs isolated at E11.5. ( C ) Fluorescence microscopy images of N-terminally FLAG-tagged and C-terminally fused with 2A-eGFP versions of ADAR1 (p110, p110 Δ7-9 , p150 and p150 Δ7-9 ) transfected into HeLa cells. DAPI shows nuclear DNA and FLAG-tagged fusions are visualized in the mCherry channel. ( D ) Western blot analysis of whole-cell extracts of HeLa cells transfected as in (C) using FLAG antibody to visualize fusion proteins (upper panel). eGFP was used for normalization (lower panel). ( E ) Detection of transfected constructs used in (C) with monoclonal anti-ADAR1 antibody 15.8.6 by western blotting. ( F ) Detection of ADAR1 in wt and ADAR1 Δ7-9 -derived MEFs after IFN induction using a polyclonal antibody directed ADAR1 p150. ( G ) Detection of ADAR1 in wt and ADAR1 Δ7-9 -derived MEFs after IFN induction using a monoclonal pan-anti ADAR1 antibody. Arrowhead marks size of predicted p150 Δ7-9 protein and asterisk depicts the size of the p110 Δ7-9 protein. ( H ) Detection of ADAR1 in wt and ADAR1 Δ7-9 brain lysates. tubulin marks detection of the lower part of the blot with an anti-tubulin antibody. (I) Quantification of ADAR p110 in three independent blots of cell lysates indicates ∼20% expression of p110 Δ7-9 protein

Article Snippet: This was done using an antibody against the p150 version of ADAR1 (Synaptic Systems) (Figure ) or by using a pan-ADAR1 antibody (15.8.6 / Santa Cruz) (Figure ).

Techniques: RNA Binding Assay, Binding Assay, Reverse Transcription Polymerase Chain Reaction, Isolation, Fluorescence, Microscopy, Transfection, Western Blot, Construct, Derivative Assay, Expressing

Journal: Nucleic Acids Research

Article Title: An internal deletion of ADAR rescued by MAVS deficiency leads to a minute phenotype

doi: 10.1093/nar/gkaa025

Figure Lengend Snippet: Phenotypic analysis of Adar Δ7-9 ; Mavs −/− mice at P15. ( A ) Dot plot showing reduced body weight of Adar Δ7-9 ; Mavs −/− compared to Adar +/+ ; Mavs −/− mice ( n = 11 per genotype). ( B ) Normalization of spleen (left) and kidney (right) weight to total body weight. A specific effect is seen on spleen, but not the kidney of Adar Δ7-9 ; Mavs −/− mice (n = 7 Adar +/+ ; Mavs −/− and n = 9 Adar Δ7-9 ; Mavs −/− ). ( C ) Dot plots showing reduced splenocytes (left, n = 6/genotype) and bone marrow cells (right, n = 8/genotype) in Adar Δ7-9 ; Mavs −/− compared to Adar +/+ ; Mavs −/− mice. Horizontal bars in (A) – (C) represent the mean; *** P < 0.001; ns = non-significant as determined by unpaired Student's t test. ( D ) Three mice per genotype were analyzed histologically at day 15 post-partum. Analyzed organs were spleen, small intestine, colon and kidney. Note the diminished cellularity of the spleen and the underdeveloped germinal center (dotted circle) in Adar Δ7-9 ; Mavs −/− mice. In the small intestine and to a lower extent in the colon, goblet cell (denoted by arrowhead) number is reduced in in Adar Δ7-9 ; Mavs −/− mice. Shown are representative sections in 8× magnification (20× in inlays) of spleen, small intestine and colon and 2× magnification (8× in inlays) of kidney with scale bars indicated.

Article Snippet: This was done using an antibody against the p150 version of ADAR1 (Synaptic Systems) (Figure ) or by using a pan-ADAR1 antibody (15.8.6 / Santa Cruz) (Figure ).

Techniques:

Journal: Nucleic Acids Research

Article Title: An internal deletion of ADAR rescued by MAVS deficiency leads to a minute phenotype

doi: 10.1093/nar/gkaa025

Figure Lengend Snippet: Altered B cell and neutrophil numbers in P15 Adar Δ7-9 ; Mavs −/− mice. ( A and B ) Immature and mature B cells in bone marrow (upper panels) and spleen (lower panels) were analyzed by flow cytometry. (A) Representative flow plots of bone marrow (upper panel) and spleen (lower panel) from Adar1 +/+ ; Mavs −/− (left panels) and Adar Δ7-9 ; Mavs −/− (right panels) animals, subgated for living NK1.1 − cells. Numbers next to the outlined areas indicate percentages within this population. (B) Dot plots showing mature (B220 + CD19 + ) and immature (B220 + CD19 − ) B cells in the bone marrow (upper panel) and spleen (lower panel). Percentages of living cells (left panels) were used to determine total cell numbers (right panels). ( C and D ) Neutrophils and inflammatory monocytes were analyzed in bone marrow and spleen of Adar1 +/+ ; Mavs −/− and Adar Δ7-9 ; Mavs −/− animals. (C) Representative flow plots of subgated for living, CD11b + cells. Numbers next to the outlined areas indicate percentages within the CD11b + population. (D) Dot plots showing neutrophils (Ly6G + Ly6C lo ) and inflammatory monocytes (Ly6G − Ly6C hi ). Percentages of living cells (left panel) were used to determine total cell numbers (right panel). n = 3 mice/genotype; horizontal bars in (C) and (D) represent the mean; * P < 0.05, ** P < 0.01 and *** P < 0.001 determined by unpaired Student's t test.

Article Snippet: This was done using an antibody against the p150 version of ADAR1 (Synaptic Systems) (Figure ) or by using a pan-ADAR1 antibody (15.8.6 / Santa Cruz) (Figure ).

Techniques: Flow Cytometry

Journal: Nucleic Acids Research

Article Title: An internal deletion of ADAR rescued by MAVS deficiency leads to a minute phenotype

doi: 10.1093/nar/gkaa025

Figure Lengend Snippet: Increased apoptosis in B cells and neutrophils in P15 Adar Δ7-9 ; Mavs −/− mice. The rate of apoptotic B cells and neutrophils was determined by flow cytometric analysis of Annexin-V and 7-AAD stained cells. ( A ) Bone marrow cells were stained and subgated for neutrophils (CD11b + Ly6G + Ly6C lo , upper panels) and B cells (CD19 + B220 + ). Left panels show representative flow plots. Numbers within the quadrants indicate percentage within the population. Right dot plots show percentages of early apoptotic (Annexin-V + 7-AAD − ), late apoptotic (Annexin-V + 7-AAD + ) and the sum of both. ( B ) Analysis of splenocytes performed as described in (A). n = 3 mice/genotype; horizontal bars in dot plots represent the mean; * P < 0.05 and ** P < 0.01 determined by unpaired Student's t test.

Article Snippet: This was done using an antibody against the p150 version of ADAR1 (Synaptic Systems) (Figure ) or by using a pan-ADAR1 antibody (15.8.6 / Santa Cruz) (Figure ).

Techniques: Staining

Journal: Nucleic Acids Research

Article Title: An internal deletion of ADAR rescued by MAVS deficiency leads to a minute phenotype

doi: 10.1093/nar/gkaa025

Figure Lengend Snippet: Differentially expressed genes of Adar Δ7-9 ; Mavs −/− liver, bone marrow and cortex. ( A ) Volcano plots showing differentially expressed genes in liver, bone marrow and cortex of P15 mice. Genes with significantly altered representation(p-value < 0.001) are marked in color. ( B ) Go-terms enriched for significant differentially expressed genes for liver and bone-marrow. ( C ) Common differentially expressed genes of liver, bone-marrow and cortex.

Article Snippet: This was done using an antibody against the p150 version of ADAR1 (Synaptic Systems) (Figure ) or by using a pan-ADAR1 antibody (15.8.6 / Santa Cruz) (Figure ).

Techniques:

Journal: Nucleic Acids Research

Article Title: An internal deletion of ADAR rescued by MAVS deficiency leads to a minute phenotype

doi: 10.1093/nar/gkaa025

Figure Lengend Snippet: Adar Δ7-9 ; Mavs −/− show altered ratios of 40S and 60S ribosomal sub-units. ( A ) Polysome profiling of P15 Adar Δ7-9 ; Mavs −/− and Mavs −/− liver (upper). RNA from fraction 7–12 loaded on a gel was stained with EtBr to confirm accumulation of 60S particles (lower). ( B ) Polysome profiling of HEK293T cells over-expressing Flag-Rps3a3 compared to mock transfected cells (upper). RNA from fraction 14–23 loaded on EtBr stained gel to confirm ribosomal peaks (lower). ( C ) Western blot of selected fractions from (B) showing incorporation of Flag-Rps3a3 in ribosomal subunits. ( D ) Expression of the pseudogene, Rps3a3 in wildtype mice in regions of craniofacial tissue at E8.5, E9.5 and E10.5 Data from .

Article Snippet: This was done using an antibody against the p150 version of ADAR1 (Synaptic Systems) (Figure ) or by using a pan-ADAR1 antibody (15.8.6 / Santa Cruz) (Figure ).

Techniques: Staining, Expressing, Transfection, Western Blot

Journal: Nucleic Acids Research

Article Title: An internal deletion of ADAR rescued by MAVS deficiency leads to a minute phenotype

doi: 10.1093/nar/gkaa025

Figure Lengend Snippet: Read coverage over Rps3a1 and Rps3a3 in rescued Adar Δ7-9 , Adar Δ2-13 and Adar E861A/E861A RNA-Seq read coverage over the UTR and coding regions of Rps3a1 and Rps3a3 in livers of P15 Adar Δ7-9 ; Mavs −/− and Mavs −/− , in E15.5 Adar1 Δ2-13 ; Mavs −/− , E15.5 Adar1 +/+ ; Mavs −/− , E11.5 Adar1 Δ2-13 ; Mavs +/+ , adult Adar1 E861A/E861A ; Ifih1 −/− and adult Adar1 +/+ ; Ifih1 −/− . Red lines in the graphs indicate the mismatches between Rps3a1 and Rps3a3 .

Article Snippet: This was done using an antibody against the p150 version of ADAR1 (Synaptic Systems) (Figure ) or by using a pan-ADAR1 antibody (15.8.6 / Santa Cruz) (Figure ).

Techniques: RNA Sequencing