Journal: Nucleic Acids Research

Article Title: Multi-layered chromatin proteomics identifies cell vulnerabilities in DNA repair

doi: 10.1093/nar/gkac1264

Figure Lengend Snippet: Reagents used in this study

Article Snippet: Blocking and incubation with primary antibodies against γH2AX (Millipore, 05–636) and THRAP3 (Novus Biologicals, NB100-40848) were performed overnight in a humidified chamber.

Techniques: Recombinant, Magnetic Beads, Plasmid Preparation

Journal: Nucleic Acids Research

Article Title: Multi-layered chromatin proteomics identifies cell vulnerabilities in DNA repair

doi: 10.1093/nar/gkac1264

Figure Lengend Snippet: IR elicits functional interaction between THRAP3 and γH2AX, while ATAD2 and TPX2 mediate DSB repair through HR. ( A ) Immunofluorescence (top panel) and proximity ligation assay (PLA, bottom panel) validation of DSB-mediated interaction between THRAP3 and γH2AX. Boxplot shows the quantification of PLA assay in untreated cells (UT) and cells exposed to ionizing radiation (IR, 5 Gy, 1 h recovery). ( B ) Deregulation of ATAD2 (top) or TPX2 (bottom) RNA level in tumor samples (red) compared to normal tissues (blue) from TCGA expressed as log2 RNAseq by Expectation-Maximization (or RSEM). ( C ) Survival probability of patients affected by Kidney renal papillary cell carcinoma (KIRP) with low (green) or high (pink) ATAD2 levels. ( D ) Number of γH2AX foci per nucleus in AID-DIvA cells left untreated or subjected to DSBs induction and repair (tamoxifen/+4OHT and Auxin, respectively), after knockdown for THRAP3, ATAD2 or TPX2 in comparison with on-target non-targeting silencing control (siCtr). Significance over siCtr is shown. ( E ) Quantification of HR (green) and NHEJ (red) repair events in U2OS-TLR cells depleted for THRAP3, ATAD2, or TPX2 and normalized over non-targeting silencing control (siCtr). Green and red asterisks reflect significant regulation in HR and NHEJ, respectively, in respect to siCtr. ( F ) Quantification of CFA in U2OS cells depleted of either ATAD2 (light green) or TPX2 (light blue) and subjected to PARP inhibitor (PARPi) alone or in combination with etoposide to promote DSBs formation. Mean values of three biological replicates normalized on untreated (UT) with standard deviations are shown. Quantification of RAD51 ( G ) and BRCA1 ( H ) foci in AID-DIvA cells knockdown for targets involved in either HR or pathway choice. *, ** and *** correspond to P -value < 0.05, 0.01 and 0.001 of ANOVA statistical test, respectively.

Article Snippet: Blocking and incubation with primary antibodies against γH2AX (Millipore, 05–636) and THRAP3 (Novus Biologicals, NB100-40848) were performed overnight in a humidified chamber.

Techniques: Functional Assay, Immunofluorescence, Proximity Ligation Assay, Biomarker Discovery, Knockdown, Comparison, Control

Journal: Nucleic Acids Research

Article Title: TRAP150 activates splicing in composite terminal exons

doi: 10.1093/nar/gku963

Figure Lengend Snippet: TRAP150 interacts and co-fractionates with the CPSF complex. ( A ) The empty or FLAG-TRAP150-expressing vector was transiently transfected into HEK293 cells. The anti-FLAG co-precipitated proteins were stained with SYPRO Ruby. Proteins identified by mass spectrometry are indicated; IgH and IgL represent immunoglobulin heavy and light chains, respectively. ( B ) HeLa cell lysates were fractionated by 15–40% glycerol gradient sedimentation. Fractions were manually collected from top to bottom, followed by immunoblotting using antibodies specific to the indicated RNA processing factors (left panel). Right panel shows TRAP150 in mock-treated or alkaline phosphatase (AP)-treated fraction 21. ( C ) The diagram depicts the domain structure of TRAP150 and its truncated forms (left). The hatched and gray regions represent the RS domain and the homology domain, respectively. The thick lines indicate the BCLAF1 and MLN51 homology regions. The right panel shows the result of immunoprecipitation. Full-length or truncated FLAG-TRAP150 was transiently expressed in HEK293 cells. Immunoprecipitation was performed using anti-FLAG; the precipitates were mock- (IP) or RNase A-treated followed by immunoblotting with the indicated antibodies. PAP, poly(A) polymerase.

Article Snippet: Antibodies against the FLAG epitope, hRrp6 and U2AF65 were from Sigma-Aldrich, antibodies against BCLAF1 (A300-608A), CPSF73 (A301-091A), CPSF160 (A301-580A), CstF64 (A301-092A), TRAP150 (A300-956A), Xrn1 (A300-443A) and Xrn2 (A301-102A) were from Bethyl Laboratories, anti-pol II C-terminal domain (4H8) was from Abcam, anti-poly(A) polymerase (H-300) was from Santa Cruz Biotechnology, anti-HA epitope was from Bioman Scientific and anti-α-tubulin (DM-1A) was from Thermo Scientific.

Techniques: Expressing, Plasmid Preparation, Transfection, Staining, Mass Spectrometry, Sedimentation, Western Blot, Immunoprecipitation

Journal: Nucleic Acids Research

Article Title: TRAP150 activates splicing in composite terminal exons

doi: 10.1093/nar/gku963

Figure Lengend Snippet: TRAP150 associates with U1 snRNP. ( A ) Expression vectors encoding different FLAG-tagged proteins as indicated were each transiently transfected into HEK293 cells. Input (5%) and anti-FLAG-immunoprecipitated RNAs (IP) were separated by acrylamide gel electrophoresis followed by northern blotting using specific snRNA probes. The immunoprecipitated proteins were detected using anti-FLAG (bottom). ( B ) Transfection and immunoprecipitation were performed as in panel A. Input and mock-treated (IP) or RNase A-treated co-precipitates were subjected to immunoblotting using specific antibodies as indicated. Asterisk represents the immunoglobulin heavy chain. ( C ) FLAG-tagged full-length and truncated TRAP150 proteins were each overexpressed in HEK293 cells and immunoprecipitated from cell lysates. Co-precipitated RNAs and proteins were subjected to northern blotting and immunoblotting as in panel A.

Article Snippet: Antibodies against the FLAG epitope, hRrp6 and U2AF65 were from Sigma-Aldrich, antibodies against BCLAF1 (A300-608A), CPSF73 (A301-091A), CPSF160 (A301-580A), CstF64 (A301-092A), TRAP150 (A300-956A), Xrn1 (A300-443A) and Xrn2 (A301-102A) were from Bethyl Laboratories, anti-pol II C-terminal domain (4H8) was from Abcam, anti-poly(A) polymerase (H-300) was from Santa Cruz Biotechnology, anti-HA epitope was from Bioman Scientific and anti-α-tubulin (DM-1A) was from Thermo Scientific.

Techniques: Expressing, Transfection, Immunoprecipitation, Acrylamide Gel Assay, Electrophoresis, Northern Blot, Western Blot

Journal: Nucleic Acids Research

Article Title: TRAP150 activates splicing in composite terminal exons

doi: 10.1093/nar/gku963

Figure Lengend Snippet: TRAP150 activates splicing of PCPA transcripts. ( A ) Lanes 1–6: HeLa cells were mock-transfected (Mock) or transfected with siRNA targeting luciferase (si-Luc.), low-GC control siRNA (si-Con.), or siRNA targeting TRAP150 (si-TRAP150), BCLAF1 (si-BCLAF1) or both (si-TRAP150/BCLAF1). Lanes 7 and 8: cells were transfected with the empty or FLAG-TRAP150-expressing vector. The PCPA transcript of NR3C1 was detected by 3′ RACE. NR3C1 mRNA was detected by primers targeted to exons 2 and 3 (Ex2–Ex3), and GAPDH served as the loading control. Knockdown of TRAP150 and BCLAF1 was evaluated by immunoblotting using specific antibody to each. Tubulin served as the control. The bar graph shows the relative expression level of the mRNA and PCPA transcripts (lanes 1–6, above) that were determined by qPCR; the expression level of each transcript was normalized to the mock control; the average and standard deviation were obtained from three independent experiments. ( B ) Transfection was as in lanes 7 and 8 of panel A. The PCPA and CSPP transcripts of NR3C1 , SEMA3C and STK17A were detected by 3′ RACE using the appropriate primers (Supplementary Table S1). Diagrams show the position of the polyadenylation signals (pAs) and primary cryptic 3′ splice sites in the PCPA cis- element-containing intron. Primers used for CSPP transcript detection are indicated by arrowheads and the size of each amplicon is shown in parenthesis. ( C ) Analysis of the selected gene that may undergo PCPA (see Materials and Methods) was essentially performed as in panel B. A representative result is shown in panels B and C; similar results were observed in at least three independent experiments and the asterisk depicts the CSPP transcripts.

Article Snippet: Antibodies against the FLAG epitope, hRrp6 and U2AF65 were from Sigma-Aldrich, antibodies against BCLAF1 (A300-608A), CPSF73 (A301-091A), CPSF160 (A301-580A), CstF64 (A301-092A), TRAP150 (A300-956A), Xrn1 (A300-443A) and Xrn2 (A301-102A) were from Bethyl Laboratories, anti-pol II C-terminal domain (4H8) was from Abcam, anti-poly(A) polymerase (H-300) was from Santa Cruz Biotechnology, anti-HA epitope was from Bioman Scientific and anti-α-tubulin (DM-1A) was from Thermo Scientific.

Techniques: Transfection, Luciferase, Control, Expressing, Plasmid Preparation, Knockdown, Western Blot, Standard Deviation, Amplification

Journal: Nucleic Acids Research

Article Title: TRAP150 activates splicing in composite terminal exons

doi: 10.1093/nar/gku963

Figure Lengend Snippet: TRAP150 forces cryptic 3′ spice site activation in PCPA transcripts. ( A ) The empty or FLAG-tagged TRAP150 or SRm160 expression vector was transiently transfected in HEK293 cells. PCPA and CSPP of NR3C1 were detected by 3′ RACE using primer sets as illustrated (top). Amplicons of primer set 1 and set 2 represent the PCPA/CSPP and read-through transcripts, respectively. Immunoblotting was performed using anti-FLAG (bottom). ( B ) The experiment was performed as in panel A, except that full-length or truncated TRAP150 was transiently expressed, and only primer set 1 was used for 3′ RACE. ( C ) The diagram (left) shows the NR3C1 minigene and positions of the 5′ splice site and polyadenylation signal mutation. The cryptic 3′ splice sites (c3′ss) and their positions relative to the 5′ splice site are indicated. The pCEP4 empty vector or HA-TRAP150 expression vector was co-transfecteand with the wild-type (WT) or a mutant NR3C1 minigene (5′ssm, pAm, 3′ss-1m or 3′ss-2m) into HeLa cells. 3′ RACE was performed as in panel B but using a vector-specific primer for the first round of PCR amplification (right). The splicing efficiency (% of CSPP) in panels B and C was calculated by relative CSPP expression levels (CSPP/CSPP+PCPA) from three independent experiments.

Article Snippet: Antibodies against the FLAG epitope, hRrp6 and U2AF65 were from Sigma-Aldrich, antibodies against BCLAF1 (A300-608A), CPSF73 (A301-091A), CPSF160 (A301-580A), CstF64 (A301-092A), TRAP150 (A300-956A), Xrn1 (A300-443A) and Xrn2 (A301-102A) were from Bethyl Laboratories, anti-pol II C-terminal domain (4H8) was from Abcam, anti-poly(A) polymerase (H-300) was from Santa Cruz Biotechnology, anti-HA epitope was from Bioman Scientific and anti-α-tubulin (DM-1A) was from Thermo Scientific.

Techniques: Activation Assay, Expressing, Plasmid Preparation, Transfection, Western Blot, Mutagenesis, Amplification

Journal: Nucleic Acids Research

Article Title: TRAP150 activates splicing in composite terminal exons

doi: 10.1093/nar/gku963

Figure Lengend Snippet: TRAP150-induced PCPA splicing is affected by functional U1 abundance and the transcription status. ( A ) HeLa cells were transfected with the following vectors: the empty (Vector) or HA-TRAP150 expression vector, the NR3C1 minigene and the wild-type (D1) or mutant (+2C) U1 decoy expression vector. Lanes 5–7: an increasing amount of the D1 plasmid. At 24 h post-transfection, cells were treated with DMSO (vehicle, lane 9) or 1 μM PB (PB, lane 10) for 4 h. 3′ RACE (upper) and immunoblotting using anti-HA were performed. Ex2–Ex3 and GAPDH were examined as in Figure . ( B ) As in panel A, transfectants were treated with 100 μM DRB (lane 3), 10 μM CPT (lane 4) or both (lane 5) for another 4 h. ( C ) HeLa cells were transfected with the indicated siRNAs for 48 h followed by transfection with the empty or HA-TRAP150 expression vector and the NR3C1 minigene for another 24 h. Immunoblotting using specific antibodies as indicated (left) indicates the effectiveness of gene silencing by siRNA. In all three panels, the PCPA/CSPP transcripts of NR3C1 were detected using 3′ RACE as in Figure . In all panels, the bar graph shows the fold change of relative CSPP expression levels (CSPP/CSPP+PCPA); the percentage of CSPP expression of each lane was normalized to the negative control (lane 4, 2 and 6 in panels A, B and C, respectively). Average and standard deviation were obtained from three independent experiments.

Article Snippet: Antibodies against the FLAG epitope, hRrp6 and U2AF65 were from Sigma-Aldrich, antibodies against BCLAF1 (A300-608A), CPSF73 (A301-091A), CPSF160 (A301-580A), CstF64 (A301-092A), TRAP150 (A300-956A), Xrn1 (A300-443A) and Xrn2 (A301-102A) were from Bethyl Laboratories, anti-pol II C-terminal domain (4H8) was from Abcam, anti-poly(A) polymerase (H-300) was from Santa Cruz Biotechnology, anti-HA epitope was from Bioman Scientific and anti-α-tubulin (DM-1A) was from Thermo Scientific.

Techniques: Functional Assay, Transfection, Plasmid Preparation, Expressing, Mutagenesis, Western Blot, Negative Control, Standard Deviation

Journal: Nucleic Acids Research

Article Title: TRAP150 activates splicing in composite terminal exons

doi: 10.1093/nar/gku963

Figure Lengend Snippet: TRAP150 activates splicing in composite but not in authentic terminal exons. The diagrams (top) show the pcDNA-CAT(In) splicing reporters without or with the NR3C1 fragment insertion into the intron or the 3′ exon, i.e. iCAT(In) and 3′eCAT(In), respectively. The asterisk depicts the cryptic 3′ splice site of NR3C1 intron 2. The empty or HA-TRAP150 expression vector was co-transfected with a splicing reporter into HeLa cells. PCPA products derived from the reporter were detected by 3′ RACE using the forward vector-specific primer and the reverse PCPA primer (bottom). Indicated products include pre-mRNA, PCPA, CSPP (asterisk) and mRNA transcripts using the authentic (triangle) or cryptic 3′ splice sites (open and close circles) in the 3′ exon of the parental CAT(In) reporter.

Article Snippet: Antibodies against the FLAG epitope, hRrp6 and U2AF65 were from Sigma-Aldrich, antibodies against BCLAF1 (A300-608A), CPSF73 (A301-091A), CPSF160 (A301-580A), CstF64 (A301-092A), TRAP150 (A300-956A), Xrn1 (A300-443A) and Xrn2 (A301-102A) were from Bethyl Laboratories, anti-pol II C-terminal domain (4H8) was from Abcam, anti-poly(A) polymerase (H-300) was from Santa Cruz Biotechnology, anti-HA epitope was from Bioman Scientific and anti-α-tubulin (DM-1A) was from Thermo Scientific.

Techniques: Expressing, Plasmid Preparation, Transfection, Derivative Assay

Journal: Nucleic Acids Research

Article Title: TRAP150 activates splicing in composite terminal exons

doi: 10.1093/nar/gku963

Figure Lengend Snippet: A model for TRAP150-activated splicing. ( A ) TRAP150 is essential for pre-mRNA splicing, as determined by a reporter assay . TRAP150 promotes the splicing efficiency of authentic introns (solid arrow line; ), perhaps via a cross-intron bridging action, but it has less or no activity in activating cryptic 3′ splice sites in downstream exons (dashed arrow line) (this study). ( B ) TRAP150 activates cryptic splicing of prematurely terminated transcripts (composite terminal exon) that are produced by promoter-proximal cleavage/polyadenylation (solid arrow line) (this study). ( C ) TRAP150 represses upstream exon inclusion by sequestering or suppressing splicing activators (SFs) that bind to the intronic enhancers ( and Supplementary Figure S1C in this study).

Article Snippet: Antibodies against the FLAG epitope, hRrp6 and U2AF65 were from Sigma-Aldrich, antibodies against BCLAF1 (A300-608A), CPSF73 (A301-091A), CPSF160 (A301-580A), CstF64 (A301-092A), TRAP150 (A300-956A), Xrn1 (A300-443A) and Xrn2 (A301-102A) were from Bethyl Laboratories, anti-pol II C-terminal domain (4H8) was from Abcam, anti-poly(A) polymerase (H-300) was from Santa Cruz Biotechnology, anti-HA epitope was from Bioman Scientific and anti-α-tubulin (DM-1A) was from Thermo Scientific.

Techniques: Reporter Assay, Activity Assay, Produced

Journal: Communications Biology

Article Title: NLRP7 maintains the genomic stability during early human embryogenesis via mediating alternative splicing

doi: 10.1038/s42003-025-07571-5

Figure Lengend Snippet: A KEGG pathway enrichment analysis of genes affected by NLRP7 mutation in hiPSCs. The RNA-seq data was derived from the study of Alici-Garipcan et al. . B Representative western blot analysis of NLRP7, γH2A.X, PARP1, PRPF8, THRAP3 and DDX39B in p-WT and p-NLRP7 −/− . C Representative immunofluorescent images of p-WT and p-NLRP7 −/− for the DNA damage marker γH2A.X (green). The nuclei were counterstained with DAPI and are shown in blue. Increased γH2A.X + cells are detected in p-NLRP7 −/− . Scale bar, 50 μm. BOX, Scale bar, 10 μm. D Quantification of the γH2A.X foci in the immunofluorescent staining shown in ( C ). Non-parametric test. E Comet assay reveals severe DNA damage in the p-NLRP7 −/− . Genomic DNA was stained with DAPI and imaged after single-cell electrophoresis. More than 200 cells were analyzed in each group. F Quantification for the length of the tail movement. Data are shown as mean ± SEM. ** P < 0.001, Non-parametric test. G Representative immunofluorescent images of p-WT and p-NLRP7 −/− for the apoptosis marker active caspase 3 (Red). Scale bar, 50 μm. BOX, Scale bar, 10 μm. H Quantification of the Active Caspase 3 in the immunofluorescent staining shown in ( G ). Data are shown as mean ± SEM. Student’s t -test.

Article Snippet: The primary antibodies and appropriate fluorohore-conjugated secondary antibodies used in this study are as follows: OCT4 (Santa Cruz, sc-5279), NANOG (Thermo Fisher Scientific, PA1-097X), NLRP7 (Abcam, ab105405), KLF17 (ATLAS ANTIBODIES, HPA024629), CDX2 (Abcam, ab76541), GATA3 (Abcam, ab199428), KRT7 (Abcam, ab68459), HLA-G (Santa Cruz, sc-21799), BrdU (BD Biosciences, 347580), γH2A.X (Cell Signaling Technology, 9718S), γH2A.X (abmart, M63324), Active-Caspase 3 (Cell Signaling Technology, 9661S), Rad51 (Abcam, ab133534), Flag (Proteintech, 66008-4-Ig), Flag (abmart, M20008S), DDX39B (Proteintech, 14798-1-AP), THRAP3 (Proteintech, 19744-1-AP), PRPF8 (Proteintech, 11171-1-AP), PARP1 (Proteintech, 66520-1-Ig), GAPDH (Proteintech, 60004-1-Ig), Goat anti-Mouse IgG (H + L) (Thermo Fisher Scientific, A-11001), and Goat anti-Rabbit IgG (H + L) (Thermo Fisher Scientific, A-11012).

Techniques: Mutagenesis, RNA Sequencing, Derivative Assay, Western Blot, Marker, Staining, Single Cell Gel Electrophoresis, Electrophoresis

Journal: Communications Biology

Article Title: NLRP7 maintains the genomic stability during early human embryogenesis via mediating alternative splicing

doi: 10.1038/s42003-025-07571-5

Figure Lengend Snippet: A Localization of NLRP7, RAD51, and DNA damage marker γH2A.X under different conditions. Scale bar, 50 μm. BOX, Scale bar, 10 μm. B Representative image of silver staining for proteins immunoprecipitated with NLRP7 using an anti-Flag antibody. The Flag-NLRP7 band is indicated with a black triangle. C KEGG analysis for proteins interacting with NLRP7 showed enriched Microbial infection and spliceosome-associated terms. D GO analysis for proteins interacting with NLRP7 showed enriched RNA processing-associated terms. E Left: Venn diagram showed overlap between the proteins that interacted with NLRP7 from this study and another independent study . Right: List of proteins in common is given. F GO analysis for proteins in common showed enriched RNA processing and RNA splicing associated terms. G–K Co-IP revealed the interaction of NLRP7 with factors related to AS and DDR, including DDX39B, PRPF8, THRAP3, and PARP1.

Article Snippet: The primary antibodies and appropriate fluorohore-conjugated secondary antibodies used in this study are as follows: OCT4 (Santa Cruz, sc-5279), NANOG (Thermo Fisher Scientific, PA1-097X), NLRP7 (Abcam, ab105405), KLF17 (ATLAS ANTIBODIES, HPA024629), CDX2 (Abcam, ab76541), GATA3 (Abcam, ab199428), KRT7 (Abcam, ab68459), HLA-G (Santa Cruz, sc-21799), BrdU (BD Biosciences, 347580), γH2A.X (Cell Signaling Technology, 9718S), γH2A.X (abmart, M63324), Active-Caspase 3 (Cell Signaling Technology, 9661S), Rad51 (Abcam, ab133534), Flag (Proteintech, 66008-4-Ig), Flag (abmart, M20008S), DDX39B (Proteintech, 14798-1-AP), THRAP3 (Proteintech, 19744-1-AP), PRPF8 (Proteintech, 11171-1-AP), PARP1 (Proteintech, 66520-1-Ig), GAPDH (Proteintech, 60004-1-Ig), Goat anti-Mouse IgG (H + L) (Thermo Fisher Scientific, A-11001), and Goat anti-Rabbit IgG (H + L) (Thermo Fisher Scientific, A-11012).

Techniques: Marker, Silver Staining, Immunoprecipitation, Infection, Co-Immunoprecipitation Assay

Journal: PLOS Pathogens

Article Title: RNA structures within Venezuelan equine encephalitis virus E1 alter macrophage replication fitness and contribute to viral emergence

doi: 10.1371/journal.ppat.1012179

Figure Lengend Snippet: (A) Top hits from dsRNA immunoprecipitation-mass spectrometry (IP-MS) of TC83 and TC83/E1 IDsyn in Raw264.7. Raw264.7 cells were infected with TC83 or TC83/E1 IDsyn at an MOI of 0.1, and viral dsRNA isolated from lysates at 24 hpi using J2 dsRNA antibody or IgG isotype control. RNA-bound proteins were identified by MS, and fold-enrichment of spectral counts relative to IgG controls was calculated. Prioritized hits were chosen based on fold enrichment scores, total spectral counts, and whether targets are known RNA binding proteins (RBPbase hits). (B) Hits equally enriched in TC83 and TC83/E1 IDsyn . (C) STRING network analysis of top proteomics hits. Candidates meeting the cutoff criteria (A) were subjected to Protein-Protein Interaction Networks Functional Enrichment Analysis. Candidate proteins identified in the screen are highlighted in red and interacting proteins in blue. (D) Enriched biological process GO terms that with a p-value >0.001, along with the observed gene count present in the STRING network (E-H) Raw264.7 were transfected with control (NSC) or pooled (3 siRNA) gene specific siRNAs targeting (E) Thrap3, (F) Fbl, (G) Ubap2l, or (H) Dhx38 for 24 hours. Cells were infected TC83 or TC83/E1 ID-syn at an MOI of 0.1, cell culture supernatants collected at 24hpi, and infectious virus titered by FFA. All siRNAs were assayed simultaneously but for visual clarity, data for each gene is shown separately along with the shared control siRNA samples. Each experiment was performed in triplicate three times independently and the mean and SD are graphed. Statistical analysis was performed using unpaired t-test. ** >0.001, ***>0.0001. Fold change and p-values are indicated on each graph.

Article Snippet: The following antibodies were used: beta-Actin Mouse mAb (Cell signaling, 8H10D10), beta-Actin Rabbit mAb (Cell signaling, 13E5), Rig-I mAb (Cell signaling, D1466), MDA-5 Rabbit mAb (Cell signaling, D74E4), Fibrillarin/U3 RNP Rabbit pAb (ABclonal, A1136), Dhx38 (ABclonal A4341), Thrap3 Rabbit pAb (ABclonal, A9396), UBAP2L (E5X4E) Rabbit mAb (Cell Signaling Technology, 40199), Goat-anti-Rabbit IRDye 800 (Licor, 926–32211), Goat-anti-mouse IRDye 680 (Licor, 926–68070).

Techniques: Immunoprecipitation, Mass Spectrometry, Protein-Protein interactions, Infection, Isolation, Control, RNA Binding Assay, Functional Assay, Transfection, Cell Culture, Virus

Journal: PLOS Pathogens

Article Title: RNA structures within Venezuelan equine encephalitis virus E1 alter macrophage replication fitness and contribute to viral emergence

doi: 10.1371/journal.ppat.1012179

Figure Lengend Snippet: (A) Schematic of translation reporter and replicon RNAs used. (B) 4μg of translation reporter RNA containing the E1 sequence from either TC83 or TC83/E1 ID-syn were nucleofected into Raw264.7 macrophages. Cell lysates were collected at indicated times post nucleofection and luciferase activity measured. Data depicted as fold change over the TC83 samples for all time points. (C) 4μg nano/firefly replicon reporter RNAs was nucleofected into Raw264.7 macrophages and cell lysates were harvested and dual-luciferase activity measured for indicated time-points. Nano-luciferase activity is displayed as fold change RLU/μg over TC83. (D-F) Raw264.7 were infected (MOI 1) with TC83 or TC83/E1 ID-syn and lysates were harvested at 1, 3, 6 and 12hpi. RT-qPCR was performed using probes specific for genomic and sub-genomic RNA species. (D) the fold change between genomic and sub-genomic RNA per time point is displayed, (E-F) viral RNA ffu equivalence was calculated using a standard from RNA derived from a known concentration virus stock. (G-H) Raw264.7 were transfected with a non-silencing control (NSC) or Thrap3 siRNA and after 24hs cells were nucleofected with 4μg of translation reporter RNA constructs. Cell lysates were collected at indicated times post nucleofection and luciferase activity measured. The fold-change in RLU/μg is displayed for each time point. Each experiment was performed in duplicate or triplicate, three times independently and the mean and SD graphed. Statistical analysis was performed using an unpaired T-test.

Article Snippet: The following antibodies were used: beta-Actin Mouse mAb (Cell signaling, 8H10D10), beta-Actin Rabbit mAb (Cell signaling, 13E5), Rig-I mAb (Cell signaling, D1466), MDA-5 Rabbit mAb (Cell signaling, D74E4), Fibrillarin/U3 RNP Rabbit pAb (ABclonal, A1136), Dhx38 (ABclonal A4341), Thrap3 Rabbit pAb (ABclonal, A9396), UBAP2L (E5X4E) Rabbit mAb (Cell Signaling Technology, 40199), Goat-anti-Rabbit IRDye 800 (Licor, 926–32211), Goat-anti-mouse IRDye 680 (Licor, 926–68070).

Techniques: Sequencing, Luciferase, Activity Assay, Infection, Quantitative RT-PCR, Derivative Assay, Concentration Assay, Virus, Transfection, Control, Construct