mouse anti adar1 monoclonal antibody  (Santa Cruz Biotechnology)

Journal: bioRxiv

Article Title: ADAR1 haploinsufficiency and sustained viral RdRp dsRNA synthesis synergize to dysregulate RNA editing and cause multi-system interferonopathy

doi: 10.1101/2025.01.21.634124

Figure Lengend Snippet: RNA isolated from brains of 4 to 5 week old WT or RdRp tg/- mice (n = 5 for WT, 6 for RdRp tg/- ) was used to measure (A) ADAR p110 and (B) ADAR p150 transcripts by qPCR. Data points represent individual animals, graphs shown means, s.d. (C) Immunoblotting for ADAR p110 and ADAR p150 in 4 to 5 week old mouse WT and RdRp tg/- brain. **** = p < 0.0001; n = 3 animals per genotype. (D) A549 cells with inducible Theiler’s virus RdRp (Tet-on system). Data are shown from two representative knockdowns. (E) RNAs isolated from the parallel knockdowns done in panel D were used for qPCR analysis to determine relative levels of the ISGs OAS and ISG15 mRNAs. mRNAs were harvested 54 hours after ADAR -targeting siRNA addition and 48 hours after dox addition. Comparisons were made between dox-treated and dox-untreated cells and expressed as fold mRNA changes induced by dox. Transfection controls (cells receiving transfection reagents but no siRNA) showed similar levels of ISG induction after dox treatment as control siRNA-transfected cells, indicating lack of contribution of transfected siRNAs to immune activation. Data are means and s.d. of triplicate biological replicates with three technical replicates each. NS: not significant; unpaired Student’s T test.

Article Snippet: For mouse tissue blots, rabbit anti-Rig-I (1:1,000 dilution, Cell Signaling, 3743), mouse anti-ADAR1 (1:500 dilution, Santa Cruz Bio, sc-73408), rabbit anti-actin (1:5,000 dilution, Cell Signaling, 4967) or the previously described tubulin antibody was used.

Techniques: Isolation, Western Blot, Virus, Transfection, Control, Activation Assay

Journal: bioRxiv

Article Title: ADAR1 haploinsufficiency and sustained viral RdRp dsRNA synthesis synergize to dysregulate RNA editing and cause multi-system interferonopathy

doi: 10.1101/2025.01.21.634124

Figure Lengend Snippet: (A) Size and coat color differences in 5-week-old, littermate RdRp tg/- and RdRp tg/- Adar +/- mice. (B) Weight differences between 4 week and 5-week-old littermate RdRp tg/- and RdRp tg/- Adar +/- mice (n = 9 and 5 respectively). (C) Dental formation changes between RdRp tg/- and RdRp tg/- Adar +/- mice. (D) H & E-stained tissue sections from kidney, brain, lung, liver, heart and spleens from RdRp tg/- and RdRp tg/- Adar +/- littermate mice. Pathological grading revealed no significant scoring in RdRp tg/- Adar +/- animals. (E) Blood Urea nitrogen from serum from 4-5 week old animals in WT, Adar +/- , RdRp tg/- , Adar +/- RdRp tg/- mice. n = 5, 6, 5, 8 for WT, Adar +/- , RdRp tg/- , Adar +/- RdRp tg/- , respectively. (F) Anti-SmAG antibodies from serum from 4-5 week old animals as measured by quantitative ELISA. Four outlier high values were seen, but two were in the WT group. n = 15, 7, 10, 9 for WT, Adar +/- , RdRp tg/- , Adar +/- RdRp tg/- , respectively. Anti-dsDNA antibody levels from matched serum was below the limit of detection for all animals tested. Where not indicated, all data and tissue sections come from a mix of male and female mice. Data in (B) was analyzed by two-way ANOVA where ****p < 0.0001. Data in (E) and (F) were analyzed by one-way ANOVA where *p < 0.05; data points represent individual animals, graphs show means and s.d.

Article Snippet: For mouse tissue blots, rabbit anti-Rig-I (1:1,000 dilution, Cell Signaling, 3743), mouse anti-ADAR1 (1:500 dilution, Santa Cruz Bio, sc-73408), rabbit anti-actin (1:5,000 dilution, Cell Signaling, 4967) or the previously described tubulin antibody was used.

Techniques: Staining, Enzyme-linked Immunosorbent Assay

Journal: bioRxiv

Article Title: ADAR1 haploinsufficiency and sustained viral RdRp dsRNA synthesis synergize to dysregulate RNA editing and cause multi-system interferonopathy

doi: 10.1101/2025.01.21.634124

Figure Lengend Snippet: Femurs from 6-week-old animals were manually de-fleshed and used for all analyses. (A) Femur lengths of WT, Adar +/- , RdRp tg/- , and RdRp tg/- Adar +/- mice (n = 8, 6, 10, and 17 animals respectively). (B) Representative µCT images, distal femur sections and whole bone. (C) Cortical bone volume fraction (BV/TV), n = 8, 7, 10, 17. (D) Trabecular number, n = 8, 7, 9, 17. (E) Trabecular separation, n = 8, 7, 9, 17. (F) Stiffness, n = 8, 7, 10, 15. (G) Maximum load, n = 8, 7, 10, 17. (H) Cortical total mineral density, n = 8, 7, 10, 17. (I) Modulus, n = 8, 7, 10, 17. (J) Ultimate stress, n = 8, 7, 10, 17. Mice with gray fur are indicated by light-blue symbols. Data were analyzed first by using a ROUT test to remove outliers (Q = 1%), which resulted in removal of two mice from one group in one panel (the RdRp tg/- Adar +/- group in , stiffness testing; hence there are 15 mice as opposed to the 17 for this genotype in the other panels). A one-way ANOVA comparing RdRp tg/- Adar +/- to each other group was used followed by a Tukey tests where * = p < 0.05, ** = p < 0.01, *** = p < 0.001, **** = p < 0.0001. Data points represent individual animals, horizontal bars indicate means.

Article Snippet: For mouse tissue blots, rabbit anti-Rig-I (1:1,000 dilution, Cell Signaling, 3743), mouse anti-ADAR1 (1:500 dilution, Santa Cruz Bio, sc-73408), rabbit anti-actin (1:5,000 dilution, Cell Signaling, 4967) or the previously described tubulin antibody was used.

Techniques:

Journal: bioRxiv

Article Title: ADAR1 haploinsufficiency and sustained viral RdRp dsRNA synthesis synergize to dysregulate RNA editing and cause multi-system interferonopathy

doi: 10.1101/2025.01.21.634124

Figure Lengend Snippet: Flash frozen brain and formalin-fixed paraffin embedded heart were collected from 6-week-old mice. (A) Heart samples were stained with Alizarin Red and calcium depositions were manually counted and shown as Alizarin Red foci per animal (n = 3, 5, 5, 10 for WT, Adar +/- , RdRp tg/ , RdRp tg/- Adar +/- , respectively). Representative heart section images are shown to the right: (i) positive control heart provided by UCDSOM Research Histology Core Laboratory; (ii) RdRp tg/- Adar +/- heart without Alizarin red foci; (iii) RdRp tg/- Adar +/- heart with a positive focus. (B) Calcium was quantified from brain tissues (n = 3, 4, 5, 5). Data were analyzed using a one-way ANOVA followed by a Tukey tests; there were no significant differences between the groups. ns: not significant.

Article Snippet: For mouse tissue blots, rabbit anti-Rig-I (1:1,000 dilution, Cell Signaling, 3743), mouse anti-ADAR1 (1:500 dilution, Santa Cruz Bio, sc-73408), rabbit anti-actin (1:5,000 dilution, Cell Signaling, 4967) or the previously described tubulin antibody was used.

Techniques: Formalin-fixed Paraffin-Embedded, Staining, Positive Control

Journal: bioRxiv

Article Title: ADAR1 haploinsufficiency and sustained viral RdRp dsRNA synthesis synergize to dysregulate RNA editing and cause multi-system interferonopathy

doi: 10.1101/2025.01.21.634124

Figure Lengend Snippet: RNA-seq from neuronal tissue from n = 4 age-matched (5 weeks) mice per group. Where possible, littermate controls were used for analysis. Two male and two female mice were used per group. (A) Multi-dimensional scaling plot of RNA expression profiles in WT-WT, Adar +/- , RdRp tg/- , and RdRp tg/- Adar +/- mice. (B) Heatmap of differentially expressed ISGs across the four genotypes. FPKM values were log transformed with 1 pseudocount to facilitate visualization. (C) Summary table of differentially expressed genes (DEGs) and proportions of which are known ISGs as determined by the Interferome database. (D) Differentially expressed genes that are shared across all group comparisons. (E) Canonical molecular pathways from IPA that are significant across group comparisons.

Article Snippet: For mouse tissue blots, rabbit anti-Rig-I (1:1,000 dilution, Cell Signaling, 3743), mouse anti-ADAR1 (1:500 dilution, Santa Cruz Bio, sc-73408), rabbit anti-actin (1:5,000 dilution, Cell Signaling, 4967) or the previously described tubulin antibody was used.

Techniques: RNA Sequencing Assay, RNA Expression, Transformation Assay

Journal: bioRxiv

Article Title: ADAR1 haploinsufficiency and sustained viral RdRp dsRNA synthesis synergize to dysregulate RNA editing and cause multi-system interferonopathy

doi: 10.1101/2025.01.21.634124

Figure Lengend Snippet: Predicted upstream signaling regulators as determined by IPA in various genotype comparisons between WT, Adar +/- , RdRp tg/- , and Adar +/- RdRp tg/- mice where red/green indicates whether the gene itself is upregulated/downregulated in the RNA-seq and orange/blue indicates predicated activation/inhibition of the molecular pathway it regulates.

Article Snippet: For mouse tissue blots, rabbit anti-Rig-I (1:1,000 dilution, Cell Signaling, 3743), mouse anti-ADAR1 (1:500 dilution, Santa Cruz Bio, sc-73408), rabbit anti-actin (1:5,000 dilution, Cell Signaling, 4967) or the previously described tubulin antibody was used.

Techniques: RNA Sequencing Assay, Activation Assay, Inhibition

Journal: bioRxiv

Article Title: ADAR1 haploinsufficiency and sustained viral RdRp dsRNA synthesis synergize to dysregulate RNA editing and cause multi-system interferonopathy

doi: 10.1101/2025.01.21.634124

Figure Lengend Snippet: (A) qPCR of three representative ISGs ( Ifit1 , Isg15 , and Oasl2 ) in brain tissue of 4-5 week old WT, Adar +/- , RdRp tg/ , and RdRp tg/- Adar +/- mice (n = 5, 5, 6, 5, respectively). (B-E) Flow cytometry analysis of cellular populations derived from the spleens of 4-5 week old mice. Single cell, RBC-lysed solutions were prepared for use in analysis. (B) BST-2 expression on the main immune cell subsets in the spleen including T cells (CD3 + ), B cells (CD19 + ), DCs (CD11c + ), granulocytes (Ly6G + ), and NK cells (NKp46 + ). n = 7, 7, 4, 4 for WT, Adar +/- , RdRp tg/ , RdRp tg/- Adar +/- , respectively. (C) Monocytes populations across all four groups. (D) BST-2 expression on monocyte/DC cell subsets including monocytes, cDC1s, cDC2s, and moDCs. (D) Activation (CD80/86 expression) of monocyte/DC subsets including monocytes, cDC1s, cDC2s, and moDCs. C-E: n = 12, 7, 10, 8 for WT, Adar +/- , RdRp tg/ , RdRp tg/- Adar +/- , respectively. For all graphs shown, data were analyzed using a one-way ANOVA followed by a Tukey tests to determine significance where * = p < 0.05, ** = p < 0.01, *** = p < 0.001, **** = p < 0.0001. Data points represent individual animals with the mean and s.d. shown as bars.

Article Snippet: For mouse tissue blots, rabbit anti-Rig-I (1:1,000 dilution, Cell Signaling, 3743), mouse anti-ADAR1 (1:500 dilution, Santa Cruz Bio, sc-73408), rabbit anti-actin (1:5,000 dilution, Cell Signaling, 4967) or the previously described tubulin antibody was used.

Techniques: Flow Cytometry, Derivative Assay, Expressing, Activation Assay

Journal: bioRxiv

Article Title: ADAR1 haploinsufficiency and sustained viral RdRp dsRNA synthesis synergize to dysregulate RNA editing and cause multi-system interferonopathy

doi: 10.1101/2025.01.21.634124

Figure Lengend Snippet: 6-week-old animals were weighed (A) and RNA was isolated from whole-brains for qPCR analysis of the ISGs Ifit1 (B) , Isg15 (C) , and Oasl2 (D) . Data were analyzed using a one-way ANOVA followed by a Tukey tests where * = p < 0.05, ** = p < 0.01, *** = p < 0.001, **** = p < 0.0001. For (A), n = 4, 5, 6, 10, and 4 for WT, Adar +/- , RdRp tg/ , RdRp tg/- Adar +/- , and RdRp tg/- Adar +/- Ifih1 -/- , respectively. For (B-D), n = 4, 3, 5, 5, and 4. Data points represent individual animals with the mean for each group shown as a bar.

Article Snippet: For mouse tissue blots, rabbit anti-Rig-I (1:1,000 dilution, Cell Signaling, 3743), mouse anti-ADAR1 (1:500 dilution, Santa Cruz Bio, sc-73408), rabbit anti-actin (1:5,000 dilution, Cell Signaling, 4967) or the previously described tubulin antibody was used.

Techniques: Isolation

Journal: bioRxiv

Article Title: ADAR1 haploinsufficiency and sustained viral RdRp dsRNA synthesis synergize to dysregulate RNA editing and cause multi-system interferonopathy

doi: 10.1101/2025.01.21.634124

Figure Lengend Snippet: Enriched diseases and biological functions across comparisons of gene expression changed between WT, Adar +/- , RdRp tg/- , and Adar +/- RdRp tg/- mice. Orange indicated that the disease or biological function is predicted to be enriched for and blue indicated is predicted to be inhibited.

Article Snippet: For mouse tissue blots, rabbit anti-Rig-I (1:1,000 dilution, Cell Signaling, 3743), mouse anti-ADAR1 (1:500 dilution, Santa Cruz Bio, sc-73408), rabbit anti-actin (1:5,000 dilution, Cell Signaling, 4967) or the previously described tubulin antibody was used.

Techniques: Expressing

Journal: bioRxiv

Article Title: ADAR1 haploinsufficiency and sustained viral RdRp dsRNA synthesis synergize to dysregulate RNA editing and cause multi-system interferonopathy

doi: 10.1101/2025.01.21.634124

Figure Lengend Snippet: Splenocytes from 4-5 week old mice were harvested and single cell, RBC-lsysed solutions were made for FACS analysis. (A) T cells, B cells, DCs, granulocytes, and NK cells show no difference across groups measured. n = 7, 7, 4, and 4 for WT, Adar +/- , RdRp tg/ , and RdRp tg/- Adar +/- , respectively. (B) DC subsets including pDCs, cDC1s, cDC2s, and moDCs show minor or no difference in populations across groups. n = 12, 7, 10, and 8 for WT, Adar +/- , RdRp tg/ , and RdRp tg/- Adar +/- , respectively. All data was analyzed using a one-way ANOVA followed by a Tukey test to determine significance. * = p < 0.05. Data represent individual animals, graphs show means and s.d.

Article Snippet: For mouse tissue blots, rabbit anti-Rig-I (1:1,000 dilution, Cell Signaling, 3743), mouse anti-ADAR1 (1:500 dilution, Santa Cruz Bio, sc-73408), rabbit anti-actin (1:5,000 dilution, Cell Signaling, 4967) or the previously described tubulin antibody was used.

Techniques:

Journal: bioRxiv

Article Title: ADAR1 haploinsufficiency and sustained viral RdRp dsRNA synthesis synergize to dysregulate RNA editing and cause multi-system interferonopathy

doi: 10.1101/2025.01.21.634124

Figure Lengend Snippet: Changes in A-to-I edits were determined via comparison of RNA-seq data with whole exome DNA sequencing. (A) Pie charts showing the proportion of editing sites within a given RNA element for each mouse genotype. (B) Table summary of the distribution of overall numbers of edited genes and sites for each genotype and the breakdown of locations of the edit sites among different RNA elements. For each of the four genotypes, four animals were sequenced and to be counted a gene must have been edited in all four animals sequenced but editing can occur anywhere in the gene. For sites, identical sites must be edited in all four animals of a genotype. (C) Venn diagrams showing overlap of edited genes (top) or sites (bottom) among the four genotypes. (D,E) Immunoblot analysis from two animals per group (D) and gene expression fold change (E) of three Adar +/- RdRp tg/- uniquely edited genes in the RNA-seq data set (as compared to WT) in 5-week-old neuronal tissue. Rig-I, Isg15, and Zbp1 were evaluated as they are each ISGs that are highly upregulated in mice expressing RdRp and are key regulators of the antiviral response. (F) Expression of Adar isoforms in neuronal tissue of 4-5 week old mice from all four genotypes, as determined by western blot (n = 2 per group).

Article Snippet: For mouse tissue blots, rabbit anti-Rig-I (1:1,000 dilution, Cell Signaling, 3743), mouse anti-ADAR1 (1:500 dilution, Santa Cruz Bio, sc-73408), rabbit anti-actin (1:5,000 dilution, Cell Signaling, 4967) or the previously described tubulin antibody was used.

Techniques: Comparison, RNA Sequencing Assay, DNA Sequencing, Western Blot, Expressing

Journal: bioRxiv

Article Title: ADAR1 haploinsufficiency and sustained viral RdRp dsRNA synthesis synergize to dysregulate RNA editing and cause multi-system interferonopathy

doi: 10.1101/2025.01.21.634124

Figure Lengend Snippet: Genes with A-to-I edit sites and RNA-seq-derived expression changes were analyzed using IPA to determine enriched diseases and biological functions across genotype comparisons (WT, Adar +/- , RdRp tg/- , and Adar +/- RdRp tg/- ). Orange indicates that the disease or biological function is predicted to be enriched for or activated and blue indicates is predicted to be inhibited.

Article Snippet: For mouse tissue blots, rabbit anti-Rig-I (1:1,000 dilution, Cell Signaling, 3743), mouse anti-ADAR1 (1:500 dilution, Santa Cruz Bio, sc-73408), rabbit anti-actin (1:5,000 dilution, Cell Signaling, 4967) or the previously described tubulin antibody was used.

Techniques: RNA Sequencing Assay, Derivative Assay, Expressing

Journal: Cell reports

Article Title: An ADAR1 dsRBD3-PKR kinase domain interaction on dsRNA inhibits PKR activation.

doi: 10.1016/j.celrep.2024.114618

Figure Lengend Snippet: Figure 2. TUNEL-positive death of intestinal crypt cells in Adar Mavs proximal intestine that begins by P8 is postponed in Adar Mavs Eif2ak2 triple mutants (A) TUNEL-positive cell death in C57BL/6 wild-type (WT), Mavs, or Adar Mavs proximal intestines at P8. White scale bar, 200 mm. (B) Quantitation of TUNEL-positive cells in proximal, medial, and distal intestines of Adar Mavs, Mavs, and C57BL/6 WT at P8. (C) TUNEL-positive cell death is postponed in Adar Mavs Eif2ak2 intestines. TUNEL-positive cell death in C57BL/6 WT, Mavs Eif2ak2, or Adar Mavs Eif2ak2 proximal intestines at P20. Scale bar, 200 mm. (D) Quantitation of TUNEL-positive cells in P20 proximal intestines (n = 3) of C57BL/6 WT, Mavs Eif2ak2 siblings, or Adar Mavs Eif2ak2. One-way ANOVA followed by Kruskal-Wallis one-way ANOVA; statistical significance is marked as ****p < 0.0001.

Article Snippet: 40 mg (intestinal tissue) of protein lysate was separated by SDS-PAGE, transferred on a nitrocellulose membrane and blotted at 95V for 1.15h at 4 C. The membranes were blocked with 5% BSA or 5% dry milk in TBS-T for 1h at RT and incubated with primary antibodies overnight at 4 C with the following dilutions: Adar1 1:200 (mouse Adar1 Santa Cruz, #sc-73408), Pkr 1:2,000 (Abcam #ab184257 [EPR19374]), p-eIF2a (Ser51) 1:1,000 (Cell Signaling, #3398), eIF2a 1:1,000 (Cell signaling, #9722), Rig-I 1:500 (Cell Signaling, [D14G6] #3743), Mda5 1:1000 (Cell Signaling [D74E4],#5321), b-actin 1:10,000 (Sigma, #A5316).

Techniques: TUNEL Assay, Quantitation Assay

Journal: Cell reports

Article Title: An ADAR1 dsRBD3-PKR kinase domain interaction on dsRNA inhibits PKR activation.

doi: 10.1016/j.celrep.2024.114618

Figure Lengend Snippet: Figure 4. Inflammation/IFN induction in Adar Mavs double mutant proximal intestines at P14 is prevented in Adar Mavs Eif2ak2 triple mutants (A) Immunoblot analysis of interferon- and PKR-related proteins in protein extracts from P14 proximal intestines of C57BL/6 wild-type (WT), Mavs, Eif2ak2, Adar Mavs, and Adar Mavs Eif2ak2 mouse pups.

Article Snippet: 40 mg (intestinal tissue) of protein lysate was separated by SDS-PAGE, transferred on a nitrocellulose membrane and blotted at 95V for 1.15h at 4 C. The membranes were blocked with 5% BSA or 5% dry milk in TBS-T for 1h at RT and incubated with primary antibodies overnight at 4 C with the following dilutions: Adar1 1:200 (mouse Adar1 Santa Cruz, #sc-73408), Pkr 1:2,000 (Abcam #ab184257 [EPR19374]), p-eIF2a (Ser51) 1:1,000 (Cell Signaling, #3398), eIF2a 1:1,000 (Cell signaling, #9722), Rig-I 1:500 (Cell Signaling, [D14G6] #3743), Mda5 1:1000 (Cell Signaling [D74E4],#5321), b-actin 1:10,000 (Sigma, #A5316).

Techniques: Mutagenesis, Western Blot

Journal: Cell reports

Article Title: An ADAR1 dsRBD3-PKR kinase domain interaction on dsRNA inhibits PKR activation.

doi: 10.1016/j.celrep.2024.114618

Figure Lengend Snippet: Figure 5. ADAR1 dsRNA binding and direct protein contact of ADAR1 dsRBD3 with PKR are both required for ADAR1-PKR interaction (A) Endogenous ADAR1p150 and ADAR1p110 iso- forms co-immunoprecipitate with endogenous PKR from A549 cells after treatment with IFN for 24 h (n = 3). Input, IP, flowthrough, and Dynabeads pro- tein G beads used for the elution are indicated. (B) AlphaFold2 prediction of the structure of full- length human PKR showing residues at the N ter- minus of helix a1 of dsRBD1 (in green, with some residue side chains in green cyan) interacting with the C lobe of the kinase domain (also in green). PKR dsRBD2 and the linkers around it have been hidden for clarity. The PKR kinase active site is at the junc- tion between the kinase domain N and C lobes, with the Thr446 residue labeled. dsRBD1 is over the loop (yellow) that is proposed to be exchanged between PKR kinase domains for autophosphorylation. (C) AlphaFold Multimer (AFM) prediction of the ADAR1 dsRBD3 (orange) protein-protein interaction with the PKR kinase domain (green) on the PKR kinase domain C lobe. ADAR1 dsRBD3 residues interacting with PKR that have been mutated are shown in magenta. Side chains of other ADAR1 dsRBD3 residues that may contact PKR are also shown in orange. These PKR dsRBD1- and ADAR1 dsRBD3-PKR kinase domain interaction models can also be viewed side by side in Video S2. (D) Endogenous PKR from A549 lung cancer cells co-immunoprecipitated with ADAR1p150-HA ex- pressed from transfected plasmids after 24 h of IFN treatment but not with ADAR1 mutant proteins lacking dsRNA binding or PKR interaction. The ADAR1 proteins tested are ADAR1p150 and p110, ADAR1 E912A catalytically inactive mutant, ADAR1 3xEAA dsRNA-binding mutant, and the ADAR1 K778E and ADAR1 R790E mutant proteins (n = 3). Flowthrough and IgG are shown in Figure S3B. (E) The ADAR2 dimer on dsRNA positions dsRBD2 (in hot pink) parallel to the dsRNA for canonical dsRBD-dsRNA contacts (see also Video S4). (F) The AFM-predicted ADAR1 dimer on dsRNA holds dsRBD3 (in orange) by the a-helical end and positions the end of dsRBD3 helix a2 close to the dsRNA. The ADAR2-dsRNA and the predicted ADAR1-dsRNA complexes can also be viewed side by side in Video S4.

Article Snippet: 40 mg (intestinal tissue) of protein lysate was separated by SDS-PAGE, transferred on a nitrocellulose membrane and blotted at 95V for 1.15h at 4 C. The membranes were blocked with 5% BSA or 5% dry milk in TBS-T for 1h at RT and incubated with primary antibodies overnight at 4 C with the following dilutions: Adar1 1:200 (mouse Adar1 Santa Cruz, #sc-73408), Pkr 1:2,000 (Abcam #ab184257 [EPR19374]), p-eIF2a (Ser51) 1:1,000 (Cell Signaling, #3398), eIF2a 1:1,000 (Cell signaling, #9722), Rig-I 1:500 (Cell Signaling, [D14G6] #3743), Mda5 1:1000 (Cell Signaling [D74E4],#5321), b-actin 1:10,000 (Sigma, #A5316).

Techniques: Binding Assay, Residue, Labeling, Immunoprecipitation, Transfection, Mutagenesis

Journal: Cell reports

Article Title: An ADAR1 dsRBD3-PKR kinase domain interaction on dsRNA inhibits PKR activation.

doi: 10.1016/j.celrep.2024.114618

Figure Lengend Snippet: Figure 6. ADAR1 dsRBD3 direct interaction with the kinase domain of PKR is required to inhibit PKR activation (A) Immunoblots showing the effects of expressed ADAR1 wild-type (WT) or mutant proteins on autophosphorylation of endogenous PKR in A549 cells treated with IFN. (B) Quantitation of p446-PKR normalized to PKR protein levels on immunoblots (n = 3). One-way ANOVA was used for statistical analysis (statistical significance is marked as *p < 0.05 and **p < 0.01). (C) Sequence chromatograms showing levels of editing at site +4 in the pri-miR 376-a2 substrate transcript expressed from a plasmid co-transfected into HEK293T cells with plasmids expressing WT or mutant ADAR1 proteins; adenosine is in green and guanosine is in black. Percentage editing is given, and the HEK293T cell editing background without additional ADAR is 17%. (D) Editing activities of expressed ADAR1p150 or mutant proteins measured via luciferase editing assay in HEK293T cells and normalized to NanoLuc expression from the same plasmid (see E). The luc dual luciferase RNA transcript has a dsRNA hairpin with an editable stop codon preceding the luciferase ORF. Unpaired t test was used for statistical significance (***p < 0.001).

Article Snippet: 40 mg (intestinal tissue) of protein lysate was separated by SDS-PAGE, transferred on a nitrocellulose membrane and blotted at 95V for 1.15h at 4 C. The membranes were blocked with 5% BSA or 5% dry milk in TBS-T for 1h at RT and incubated with primary antibodies overnight at 4 C with the following dilutions: Adar1 1:200 (mouse Adar1 Santa Cruz, #sc-73408), Pkr 1:2,000 (Abcam #ab184257 [EPR19374]), p-eIF2a (Ser51) 1:1,000 (Cell Signaling, #3398), eIF2a 1:1,000 (Cell signaling, #9722), Rig-I 1:500 (Cell Signaling, [D14G6] #3743), Mda5 1:1000 (Cell Signaling [D74E4],#5321), b-actin 1:10,000 (Sigma, #A5316).

Techniques: Activation Assay, Western Blot, Mutagenesis, Quantitation Assay, Sequencing, Plasmid Preparation, Transfection, Expressing, Luciferase

Journal: Cell reports

Article Title: An ADAR1 dsRBD3-PKR kinase domain interaction on dsRNA inhibits PKR activation.

doi: 10.1016/j.celrep.2024.114618

Figure Lengend Snippet: Figure 7. Subcellular co-localization of ADAR1 and PKR requires both ADAR1 dsRNA binding and ADAR1-PKR protein-protein interaction (A) Proximity ligation assay (PLA) in IFN-treated A549 cells expressing ADAR1p150, ADAR1p110, or mutant proteins. Fluorescent puncta indicate direct inter- action between ADAR1 wild-type/mutant proteins and endogenous PKR (within 40 mm). (B) Quantitation of fluorescent puncta per nucleus for ADAR1 wild-type and mutant proteins analyzed with the Particle analysis plugin in ImageJ. Unpaired t test was used for statistical analysis (****p < 0.0001). (C) Cartoon showing how different domains of the proteins behave during dsRNA activation of PKR, on the right, and how ADAR1-PKR protein-protein interaction can still prevent PKR activation following dsRNA binding.

Article Snippet: 40 mg (intestinal tissue) of protein lysate was separated by SDS-PAGE, transferred on a nitrocellulose membrane and blotted at 95V for 1.15h at 4 C. The membranes were blocked with 5% BSA or 5% dry milk in TBS-T for 1h at RT and incubated with primary antibodies overnight at 4 C with the following dilutions: Adar1 1:200 (mouse Adar1 Santa Cruz, #sc-73408), Pkr 1:2,000 (Abcam #ab184257 [EPR19374]), p-eIF2a (Ser51) 1:1,000 (Cell Signaling, #3398), eIF2a 1:1,000 (Cell signaling, #9722), Rig-I 1:500 (Cell Signaling, [D14G6] #3743), Mda5 1:1000 (Cell Signaling [D74E4],#5321), b-actin 1:10,000 (Sigma, #A5316).

Techniques: Binding Assay, Proximity Ligation Assay, Expressing, Mutagenesis, Quantitation Assay, Particle Size Analysis, Activation Assay

Journal: Cell reports

Article Title: An ADAR1 dsRBD3-PKR kinase domain interaction on dsRNA inhibits PKR activation.

doi: 10.1016/j.celrep.2024.114618

Figure Lengend Snippet: Figure 2. TUNEL-positive death of intestinal crypt cells in Adar Mavs proximal intestine that begins by P8 is postponed in Adar Mavs Eif2ak2 triple mutants (A) TUNEL-positive cell death in C57BL/6 wild-type (WT), Mavs, or Adar Mavs proximal intestines at P8. White scale bar, 200 mm. (B) Quantitation of TUNEL-positive cells in proximal, medial, and distal intestines of Adar Mavs, Mavs, and C57BL/6 WT at P8. (C) TUNEL-positive cell death is postponed in Adar Mavs Eif2ak2 intestines. TUNEL-positive cell death in C57BL/6 WT, Mavs Eif2ak2, or Adar Mavs Eif2ak2 proximal intestines at P20. Scale bar, 200 mm. (D) Quantitation of TUNEL-positive cells in P20 proximal intestines (n = 3) of C57BL/6 WT, Mavs Eif2ak2 siblings, or Adar Mavs Eif2ak2. One-way ANOVA followed by Kruskal-Wallis one-way ANOVA; statistical significance is marked as ****p < 0.0001.

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies Mouse monoclonal Anti-Adar1 antibody Santa Cruz Biotechnology #sc-73408, RRID:AB_2222767 Anti-hADAR1 Antibodies-online.com #ABIN2855100 Recombinant Anti-PKR antibody [EPR19374] Abcam #ab184257, RRID:AB_2916271 Phospho-eIF2alpha (Ser51) (D9G8) XP Rabbit mAb Cell Signaling #3398, RRID:AB_2096481 eIF2a Cell Signaling #9722, RRID:AB_2230924 Rabbit Anti-Rig-I Monoclonal Antibody [D14G6] Cell Signaling #3743, RRID:AB_2269233 Mda5 [D74E4] Rabbit mAb Cell Signaling #5321, RRID:AB_10694490 Mouse b-Actin Sigma-Aldrich #A5441, RRID:AB_476744 ADAR1 Antibodies-online.com #ABIN2855100 HA-Tag (C29F4) Rabbit mAb Cell signaling #3724, RRID:AB_1549585 HA tag Mouse antibodies.com #A85278, RRID:AB_2748877 HA tag (6E2) Mouse mAb Cell signaling #2367, RRID:AB_10691311 Goat anti-Mouse Thermo Fisher #31430 Anti-Rabbit IgG Merck #A0545 Rabbit Polyclonal Anti-GFAP Dako-Agilent #ZO334 Rabbit mAb beta Tubulin [EP1569Y] Abcam #ab52623 RRID:AB_869991 Donkey anti-Rabbit IgG Alexa Fluor 555 Invitrogen #AB_162543 Donkey Anti-Rat IgG Cy3 Jackson #AB_2340667 Rabbit mAb Olfm4 (D6Y5A) Cell Signaling #39141, RRID:AB_2650511 Goat anti-Rabbit IgG Alexa Fluor 488 Invitrogen A11034 Rabbit anti-Ki-67 Zytomed systems #RBK027 SignalStain Boost IHC Detection Reagent Cell Signaling #8114, RRID:AB_10544930 Normal Rabbit IgG Cell Signaling #2729 RRID:AB_1031062 Bacterial and virus strains One ShotTM TOP10 Chemically Competent E. coli Thermo Fisher C404003 Chemicals, peptides, and recombinant proteins MEM media Biosera LM-E1140 DMEM media Biosera LM-D1099 FBS Merk F7524 penicillin/streptomycin Biosera XCA4122 Ambion mMESSAGE mMACHINE T7 Transcription Kit Thermo Fisher #AM1344 mirPremierTM microRNA Isolation Kit Sigma-Aldrich #SNC50 RNeasy Mini kit Qiagen #74104 EliZyme FAST Taq MIX Red PCR kit Elisabeth Pharmacon Midori Green Advance NIPPON Genetics MG04 EDTA DCS Innovative Diagnostik-Systeme #EL010C500 SignalStain DAB Substrate Cell Signaling #8059 VECTASHIELD PLUS Antifade Mounting Medium with DAPI Vector Laboratories #H-1200-10 ProLong Gold Antifade Mountant with DAPI Invitrogen P36931 Clic-it plus TUNEL assay Life Technologies #C10617 HaltTM Protease Inhibitor Cocktail, EDTA-Free (100X) Thermo Fisher #78439 HaltTM Phosphatase Inhibitor Cocktail (100X) Thermo Fisher #78427 (Continued on next page) Cell Reports 43, 114618, August 27, 2024 17

Techniques: TUNEL Assay, Quantitation Assay

Journal: Cell reports

Article Title: An ADAR1 dsRBD3-PKR kinase domain interaction on dsRNA inhibits PKR activation.

doi: 10.1016/j.celrep.2024.114618

Figure Lengend Snippet: Figure 4. Inflammation/IFN induction in Adar Mavs double mutant proximal intestines at P14 is prevented in Adar Mavs Eif2ak2 triple mutants (A) Immunoblot analysis of interferon- and PKR-related proteins in protein extracts from P14 proximal intestines of C57BL/6 wild-type (WT), Mavs, Eif2ak2, Adar Mavs, and Adar Mavs Eif2ak2 mouse pups.

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies Mouse monoclonal Anti-Adar1 antibody Santa Cruz Biotechnology #sc-73408, RRID:AB_2222767 Anti-hADAR1 Antibodies-online.com #ABIN2855100 Recombinant Anti-PKR antibody [EPR19374] Abcam #ab184257, RRID:AB_2916271 Phospho-eIF2alpha (Ser51) (D9G8) XP Rabbit mAb Cell Signaling #3398, RRID:AB_2096481 eIF2a Cell Signaling #9722, RRID:AB_2230924 Rabbit Anti-Rig-I Monoclonal Antibody [D14G6] Cell Signaling #3743, RRID:AB_2269233 Mda5 [D74E4] Rabbit mAb Cell Signaling #5321, RRID:AB_10694490 Mouse b-Actin Sigma-Aldrich #A5441, RRID:AB_476744 ADAR1 Antibodies-online.com #ABIN2855100 HA-Tag (C29F4) Rabbit mAb Cell signaling #3724, RRID:AB_1549585 HA tag Mouse antibodies.com #A85278, RRID:AB_2748877 HA tag (6E2) Mouse mAb Cell signaling #2367, RRID:AB_10691311 Goat anti-Mouse Thermo Fisher #31430 Anti-Rabbit IgG Merck #A0545 Rabbit Polyclonal Anti-GFAP Dako-Agilent #ZO334 Rabbit mAb beta Tubulin [EP1569Y] Abcam #ab52623 RRID:AB_869991 Donkey anti-Rabbit IgG Alexa Fluor 555 Invitrogen #AB_162543 Donkey Anti-Rat IgG Cy3 Jackson #AB_2340667 Rabbit mAb Olfm4 (D6Y5A) Cell Signaling #39141, RRID:AB_2650511 Goat anti-Rabbit IgG Alexa Fluor 488 Invitrogen A11034 Rabbit anti-Ki-67 Zytomed systems #RBK027 SignalStain Boost IHC Detection Reagent Cell Signaling #8114, RRID:AB_10544930 Normal Rabbit IgG Cell Signaling #2729 RRID:AB_1031062 Bacterial and virus strains One ShotTM TOP10 Chemically Competent E. coli Thermo Fisher C404003 Chemicals, peptides, and recombinant proteins MEM media Biosera LM-E1140 DMEM media Biosera LM-D1099 FBS Merk F7524 penicillin/streptomycin Biosera XCA4122 Ambion mMESSAGE mMACHINE T7 Transcription Kit Thermo Fisher #AM1344 mirPremierTM microRNA Isolation Kit Sigma-Aldrich #SNC50 RNeasy Mini kit Qiagen #74104 EliZyme FAST Taq MIX Red PCR kit Elisabeth Pharmacon Midori Green Advance NIPPON Genetics MG04 EDTA DCS Innovative Diagnostik-Systeme #EL010C500 SignalStain DAB Substrate Cell Signaling #8059 VECTASHIELD PLUS Antifade Mounting Medium with DAPI Vector Laboratories #H-1200-10 ProLong Gold Antifade Mountant with DAPI Invitrogen P36931 Clic-it plus TUNEL assay Life Technologies #C10617 HaltTM Protease Inhibitor Cocktail, EDTA-Free (100X) Thermo Fisher #78439 HaltTM Phosphatase Inhibitor Cocktail (100X) Thermo Fisher #78427 (Continued on next page) Cell Reports 43, 114618, August 27, 2024 17

Techniques: Mutagenesis, Western Blot

Journal: Cell reports

Article Title: An ADAR1 dsRBD3-PKR kinase domain interaction on dsRNA inhibits PKR activation.

doi: 10.1016/j.celrep.2024.114618

Figure Lengend Snippet: Figure 5. ADAR1 dsRNA binding and direct protein contact of ADAR1 dsRBD3 with PKR are both required for ADAR1-PKR interaction (A) Endogenous ADAR1p150 and ADAR1p110 iso- forms co-immunoprecipitate with endogenous PKR from A549 cells after treatment with IFN for 24 h (n = 3). Input, IP, flowthrough, and Dynabeads pro- tein G beads used for the elution are indicated. (B) AlphaFold2 prediction of the structure of full- length human PKR showing residues at the N ter- minus of helix a1 of dsRBD1 (in green, with some residue side chains in green cyan) interacting with the C lobe of the kinase domain (also in green). PKR dsRBD2 and the linkers around it have been hidden for clarity. The PKR kinase active site is at the junc- tion between the kinase domain N and C lobes, with the Thr446 residue labeled. dsRBD1 is over the loop (yellow) that is proposed to be exchanged between PKR kinase domains for autophosphorylation. (C) AlphaFold Multimer (AFM) prediction of the ADAR1 dsRBD3 (orange) protein-protein interaction with the PKR kinase domain (green) on the PKR kinase domain C lobe. ADAR1 dsRBD3 residues interacting with PKR that have been mutated are shown in magenta. Side chains of other ADAR1 dsRBD3 residues that may contact PKR are also shown in orange. These PKR dsRBD1- and ADAR1 dsRBD3-PKR kinase domain interaction models can also be viewed side by side in Video S2. (D) Endogenous PKR from A549 lung cancer cells co-immunoprecipitated with ADAR1p150-HA ex- pressed from transfected plasmids after 24 h of IFN treatment but not with ADAR1 mutant proteins lacking dsRNA binding or PKR interaction. The ADAR1 proteins tested are ADAR1p150 and p110, ADAR1 E912A catalytically inactive mutant, ADAR1 3xEAA dsRNA-binding mutant, and the ADAR1 K778E and ADAR1 R790E mutant proteins (n = 3). Flowthrough and IgG are shown in Figure S3B. (E) The ADAR2 dimer on dsRNA positions dsRBD2 (in hot pink) parallel to the dsRNA for canonical dsRBD-dsRNA contacts (see also Video S4). (F) The AFM-predicted ADAR1 dimer on dsRNA holds dsRBD3 (in orange) by the a-helical end and positions the end of dsRBD3 helix a2 close to the dsRNA. The ADAR2-dsRNA and the predicted ADAR1-dsRNA complexes can also be viewed side by side in Video S4.

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies Mouse monoclonal Anti-Adar1 antibody Santa Cruz Biotechnology #sc-73408, RRID:AB_2222767 Anti-hADAR1 Antibodies-online.com #ABIN2855100 Recombinant Anti-PKR antibody [EPR19374] Abcam #ab184257, RRID:AB_2916271 Phospho-eIF2alpha (Ser51) (D9G8) XP Rabbit mAb Cell Signaling #3398, RRID:AB_2096481 eIF2a Cell Signaling #9722, RRID:AB_2230924 Rabbit Anti-Rig-I Monoclonal Antibody [D14G6] Cell Signaling #3743, RRID:AB_2269233 Mda5 [D74E4] Rabbit mAb Cell Signaling #5321, RRID:AB_10694490 Mouse b-Actin Sigma-Aldrich #A5441, RRID:AB_476744 ADAR1 Antibodies-online.com #ABIN2855100 HA-Tag (C29F4) Rabbit mAb Cell signaling #3724, RRID:AB_1549585 HA tag Mouse antibodies.com #A85278, RRID:AB_2748877 HA tag (6E2) Mouse mAb Cell signaling #2367, RRID:AB_10691311 Goat anti-Mouse Thermo Fisher #31430 Anti-Rabbit IgG Merck #A0545 Rabbit Polyclonal Anti-GFAP Dako-Agilent #ZO334 Rabbit mAb beta Tubulin [EP1569Y] Abcam #ab52623 RRID:AB_869991 Donkey anti-Rabbit IgG Alexa Fluor 555 Invitrogen #AB_162543 Donkey Anti-Rat IgG Cy3 Jackson #AB_2340667 Rabbit mAb Olfm4 (D6Y5A) Cell Signaling #39141, RRID:AB_2650511 Goat anti-Rabbit IgG Alexa Fluor 488 Invitrogen A11034 Rabbit anti-Ki-67 Zytomed systems #RBK027 SignalStain Boost IHC Detection Reagent Cell Signaling #8114, RRID:AB_10544930 Normal Rabbit IgG Cell Signaling #2729 RRID:AB_1031062 Bacterial and virus strains One ShotTM TOP10 Chemically Competent E. coli Thermo Fisher C404003 Chemicals, peptides, and recombinant proteins MEM media Biosera LM-E1140 DMEM media Biosera LM-D1099 FBS Merk F7524 penicillin/streptomycin Biosera XCA4122 Ambion mMESSAGE mMACHINE T7 Transcription Kit Thermo Fisher #AM1344 mirPremierTM microRNA Isolation Kit Sigma-Aldrich #SNC50 RNeasy Mini kit Qiagen #74104 EliZyme FAST Taq MIX Red PCR kit Elisabeth Pharmacon Midori Green Advance NIPPON Genetics MG04 EDTA DCS Innovative Diagnostik-Systeme #EL010C500 SignalStain DAB Substrate Cell Signaling #8059 VECTASHIELD PLUS Antifade Mounting Medium with DAPI Vector Laboratories #H-1200-10 ProLong Gold Antifade Mountant with DAPI Invitrogen P36931 Clic-it plus TUNEL assay Life Technologies #C10617 HaltTM Protease Inhibitor Cocktail, EDTA-Free (100X) Thermo Fisher #78439 HaltTM Phosphatase Inhibitor Cocktail (100X) Thermo Fisher #78427 (Continued on next page) Cell Reports 43, 114618, August 27, 2024 17

Techniques: Binding Assay, Residue, Labeling, Immunoprecipitation, Transfection, Mutagenesis

Journal: Cell reports

Article Title: An ADAR1 dsRBD3-PKR kinase domain interaction on dsRNA inhibits PKR activation.

doi: 10.1016/j.celrep.2024.114618

Figure Lengend Snippet: Figure 6. ADAR1 dsRBD3 direct interaction with the kinase domain of PKR is required to inhibit PKR activation (A) Immunoblots showing the effects of expressed ADAR1 wild-type (WT) or mutant proteins on autophosphorylation of endogenous PKR in A549 cells treated with IFN. (B) Quantitation of p446-PKR normalized to PKR protein levels on immunoblots (n = 3). One-way ANOVA was used for statistical analysis (statistical significance is marked as *p < 0.05 and **p < 0.01). (C) Sequence chromatograms showing levels of editing at site +4 in the pri-miR 376-a2 substrate transcript expressed from a plasmid co-transfected into HEK293T cells with plasmids expressing WT or mutant ADAR1 proteins; adenosine is in green and guanosine is in black. Percentage editing is given, and the HEK293T cell editing background without additional ADAR is 17%. (D) Editing activities of expressed ADAR1p150 or mutant proteins measured via luciferase editing assay in HEK293T cells and normalized to NanoLuc expression from the same plasmid (see E). The luc dual luciferase RNA transcript has a dsRNA hairpin with an editable stop codon preceding the luciferase ORF. Unpaired t test was used for statistical significance (***p < 0.001).

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies Mouse monoclonal Anti-Adar1 antibody Santa Cruz Biotechnology #sc-73408, RRID:AB_2222767 Anti-hADAR1 Antibodies-online.com #ABIN2855100 Recombinant Anti-PKR antibody [EPR19374] Abcam #ab184257, RRID:AB_2916271 Phospho-eIF2alpha (Ser51) (D9G8) XP Rabbit mAb Cell Signaling #3398, RRID:AB_2096481 eIF2a Cell Signaling #9722, RRID:AB_2230924 Rabbit Anti-Rig-I Monoclonal Antibody [D14G6] Cell Signaling #3743, RRID:AB_2269233 Mda5 [D74E4] Rabbit mAb Cell Signaling #5321, RRID:AB_10694490 Mouse b-Actin Sigma-Aldrich #A5441, RRID:AB_476744 ADAR1 Antibodies-online.com #ABIN2855100 HA-Tag (C29F4) Rabbit mAb Cell signaling #3724, RRID:AB_1549585 HA tag Mouse antibodies.com #A85278, RRID:AB_2748877 HA tag (6E2) Mouse mAb Cell signaling #2367, RRID:AB_10691311 Goat anti-Mouse Thermo Fisher #31430 Anti-Rabbit IgG Merck #A0545 Rabbit Polyclonal Anti-GFAP Dako-Agilent #ZO334 Rabbit mAb beta Tubulin [EP1569Y] Abcam #ab52623 RRID:AB_869991 Donkey anti-Rabbit IgG Alexa Fluor 555 Invitrogen #AB_162543 Donkey Anti-Rat IgG Cy3 Jackson #AB_2340667 Rabbit mAb Olfm4 (D6Y5A) Cell Signaling #39141, RRID:AB_2650511 Goat anti-Rabbit IgG Alexa Fluor 488 Invitrogen A11034 Rabbit anti-Ki-67 Zytomed systems #RBK027 SignalStain Boost IHC Detection Reagent Cell Signaling #8114, RRID:AB_10544930 Normal Rabbit IgG Cell Signaling #2729 RRID:AB_1031062 Bacterial and virus strains One ShotTM TOP10 Chemically Competent E. coli Thermo Fisher C404003 Chemicals, peptides, and recombinant proteins MEM media Biosera LM-E1140 DMEM media Biosera LM-D1099 FBS Merk F7524 penicillin/streptomycin Biosera XCA4122 Ambion mMESSAGE mMACHINE T7 Transcription Kit Thermo Fisher #AM1344 mirPremierTM microRNA Isolation Kit Sigma-Aldrich #SNC50 RNeasy Mini kit Qiagen #74104 EliZyme FAST Taq MIX Red PCR kit Elisabeth Pharmacon Midori Green Advance NIPPON Genetics MG04 EDTA DCS Innovative Diagnostik-Systeme #EL010C500 SignalStain DAB Substrate Cell Signaling #8059 VECTASHIELD PLUS Antifade Mounting Medium with DAPI Vector Laboratories #H-1200-10 ProLong Gold Antifade Mountant with DAPI Invitrogen P36931 Clic-it plus TUNEL assay Life Technologies #C10617 HaltTM Protease Inhibitor Cocktail, EDTA-Free (100X) Thermo Fisher #78439 HaltTM Phosphatase Inhibitor Cocktail (100X) Thermo Fisher #78427 (Continued on next page) Cell Reports 43, 114618, August 27, 2024 17

Techniques: Activation Assay, Western Blot, Mutagenesis, Quantitation Assay, Sequencing, Plasmid Preparation, Transfection, Expressing, Luciferase

Journal: Cell reports

Article Title: An ADAR1 dsRBD3-PKR kinase domain interaction on dsRNA inhibits PKR activation.

doi: 10.1016/j.celrep.2024.114618

Figure Lengend Snippet: Figure 7. Subcellular co-localization of ADAR1 and PKR requires both ADAR1 dsRNA binding and ADAR1-PKR protein-protein interaction (A) Proximity ligation assay (PLA) in IFN-treated A549 cells expressing ADAR1p150, ADAR1p110, or mutant proteins. Fluorescent puncta indicate direct inter- action between ADAR1 wild-type/mutant proteins and endogenous PKR (within 40 mm). (B) Quantitation of fluorescent puncta per nucleus for ADAR1 wild-type and mutant proteins analyzed with the Particle analysis plugin in ImageJ. Unpaired t test was used for statistical analysis (****p < 0.0001). (C) Cartoon showing how different domains of the proteins behave during dsRNA activation of PKR, on the right, and how ADAR1-PKR protein-protein interaction can still prevent PKR activation following dsRNA binding.

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies Mouse monoclonal Anti-Adar1 antibody Santa Cruz Biotechnology #sc-73408, RRID:AB_2222767 Anti-hADAR1 Antibodies-online.com #ABIN2855100 Recombinant Anti-PKR antibody [EPR19374] Abcam #ab184257, RRID:AB_2916271 Phospho-eIF2alpha (Ser51) (D9G8) XP Rabbit mAb Cell Signaling #3398, RRID:AB_2096481 eIF2a Cell Signaling #9722, RRID:AB_2230924 Rabbit Anti-Rig-I Monoclonal Antibody [D14G6] Cell Signaling #3743, RRID:AB_2269233 Mda5 [D74E4] Rabbit mAb Cell Signaling #5321, RRID:AB_10694490 Mouse b-Actin Sigma-Aldrich #A5441, RRID:AB_476744 ADAR1 Antibodies-online.com #ABIN2855100 HA-Tag (C29F4) Rabbit mAb Cell signaling #3724, RRID:AB_1549585 HA tag Mouse antibodies.com #A85278, RRID:AB_2748877 HA tag (6E2) Mouse mAb Cell signaling #2367, RRID:AB_10691311 Goat anti-Mouse Thermo Fisher #31430 Anti-Rabbit IgG Merck #A0545 Rabbit Polyclonal Anti-GFAP Dako-Agilent #ZO334 Rabbit mAb beta Tubulin [EP1569Y] Abcam #ab52623 RRID:AB_869991 Donkey anti-Rabbit IgG Alexa Fluor 555 Invitrogen #AB_162543 Donkey Anti-Rat IgG Cy3 Jackson #AB_2340667 Rabbit mAb Olfm4 (D6Y5A) Cell Signaling #39141, RRID:AB_2650511 Goat anti-Rabbit IgG Alexa Fluor 488 Invitrogen A11034 Rabbit anti-Ki-67 Zytomed systems #RBK027 SignalStain Boost IHC Detection Reagent Cell Signaling #8114, RRID:AB_10544930 Normal Rabbit IgG Cell Signaling #2729 RRID:AB_1031062 Bacterial and virus strains One ShotTM TOP10 Chemically Competent E. coli Thermo Fisher C404003 Chemicals, peptides, and recombinant proteins MEM media Biosera LM-E1140 DMEM media Biosera LM-D1099 FBS Merk F7524 penicillin/streptomycin Biosera XCA4122 Ambion mMESSAGE mMACHINE T7 Transcription Kit Thermo Fisher #AM1344 mirPremierTM microRNA Isolation Kit Sigma-Aldrich #SNC50 RNeasy Mini kit Qiagen #74104 EliZyme FAST Taq MIX Red PCR kit Elisabeth Pharmacon Midori Green Advance NIPPON Genetics MG04 EDTA DCS Innovative Diagnostik-Systeme #EL010C500 SignalStain DAB Substrate Cell Signaling #8059 VECTASHIELD PLUS Antifade Mounting Medium with DAPI Vector Laboratories #H-1200-10 ProLong Gold Antifade Mountant with DAPI Invitrogen P36931 Clic-it plus TUNEL assay Life Technologies #C10617 HaltTM Protease Inhibitor Cocktail, EDTA-Free (100X) Thermo Fisher #78439 HaltTM Phosphatase Inhibitor Cocktail (100X) Thermo Fisher #78427 (Continued on next page) Cell Reports 43, 114618, August 27, 2024 17

Techniques: Binding Assay, Proximity Ligation Assay, Expressing, Mutagenesis, Quantitation Assay, Particle Size Analysis, Activation Assay