p426 72q gpd plasmid  (Addgene inc)

Journal: Disease Models & Mechanisms

Article Title: Integrated multi-omics analysis of Huntington disease identifies pathways that modulate protein aggregation

doi: 10.1242/dmm.049492

Figure Lengend Snippet: Metabolomic study using an HD yeast model. (A-F) Heat maps of targeted metabolomics in Htt yeast models 46Q, 72Q and 103Q compared to that of 25Q in positive and negative modes. Altered metabolites in 46Q compared to 25Q in positive (A) and negative (B) mode. Altered metabolites in 72Q compared to 25Q in positive (C) and negative mode (D). (E) Altered metabolites in 103Q compared to 25Q in positive (E) and negative (F) mode.

Article Snippet: A total of 8×10 8 cells was taken for transformation with p426 25Q GPD, p426 46Q GPD, p426 72Q GPD and p426 103Q GPD (Addgene plasmids #1181 , #1182 , #1183 and #1184 , respectively).

Techniques:

Journal: Disease Models & Mechanisms

Article Title: Integrated multi-omics analysis of Huntington disease identifies pathways that modulate protein aggregation

doi: 10.1242/dmm.049492

Figure Lengend Snippet: Metabolite set enrichment analysis (MESA) and shared deregulated pathways. (A) Yeast metabolomics (present study) show significantly different levels of metabolites in yeasts 46Q, 72Q and 103Q compared to 25Q. (B) MSEA showing shared pathways deregulated in 25Q vs 46Q, 25Q vs 72Q and 25Q vs 103Q. (C) Metabolomics pathway overlaps determined in our study (yeasts 25Q vs 46Q, 25Q vs 72Q and 25Q vs 103Q) and literature show pathways commonly deregulated in yeast 103Q compared to yeast 25Q. (D,E) Comparative analysis of significantly enriched metabolic pathways identified in a HD mouse model (D) and in HD patients (E). (F) Metabolomics of previously published deregulated metabolic pathways in human (literature) and deregulated pathways identified in our HD patient cohort (present study), showing an overlap of three deregulated pathways. (G) Plotted is the overlap of deregulated metabolic pathways identified in yeast and mouse HD models, and HD patients. BS, brain stem; CB, cerebellum; CSF, cerebrospinal fluid; CT, cortex; FL, frontal lobe; ST, striatum.

Article Snippet: A total of 8×10 8 cells was taken for transformation with p426 25Q GPD, p426 46Q GPD, p426 72Q GPD and p426 103Q GPD (Addgene plasmids #1181 , #1182 , #1183 and #1184 , respectively).

Techniques:

Journal: Scientific Reports

Article Title: Identification of an XRCC1 DNA binding activity essential for retention at sites of DNA damage

doi: 10.1038/s41598-019-39543-1

Figure Lengend Snippet: Recruitment of XRCC1 and XRCC1-P1/3 mutant to sites of micro-irradiation damage. ( a ) Time course of XRCC1 DNA damage localization 20–120 seconds post-irradiation. ( b ) Relative levels of XRCC1 and XRCC1-P1/3 mutant localization over 120 seconds post-irradiation. ( c ) Time course of XRCC1 DNA damage localization 2–30 min post-irradiation. ( d ) Comparison of number of cells showing detectable localization of XRCC1 at damage sites over 62 min following irradiation. Blue and orange bars represent wild type and P1/3 mutant XRCC1, respectively. Scale bars, 5 µm.

Article Snippet: Primers used in PCR reactions for vector construction are listed in Supplementary Fig. . PCR products were first moved into a pDONR201 entry vector (Invitrogen) and subsequently recombined into destination vectors, pDEST15 (Invitrogen) for full length XRCC1; pDEST544 (Addgene 11519) for XRCC1 219–415 and XRCC1 219–300 ; and pDEST17 (Invitrogen) for XRCC1 1–183 and XRCC1 301–415 .

Techniques: Mutagenesis, Irradiation

Journal: Scientific Reports

Article Title: Identification of an XRCC1 DNA binding activity essential for retention at sites of DNA damage

doi: 10.1038/s41598-019-39543-1

Figure Lengend Snippet: Comparison of DNA binding activities of XRCC1 truncations. ( a ) Domain organization of XRCC1 with truncation boundaries indicated by arrows: Full length (blue), 1–183 (purple), 219–633 (green), 219–415 (red), 219–300 (orange) and 301–415 (cyan). ( b – g ) DNA binding activity of XRCC1 truncations (µM concentrations) monitored by electrophoretic mobility shift using fluorescent 39 bp duplex DNA. Discontinuous PAGE was used to facilitate band resolution. Full length gels are in Supplementary Fig. .

Article Snippet: Primers used in PCR reactions for vector construction are listed in Supplementary Fig. . PCR products were first moved into a pDONR201 entry vector (Invitrogen) and subsequently recombined into destination vectors, pDEST15 (Invitrogen) for full length XRCC1; pDEST544 (Addgene 11519) for XRCC1 219–415 and XRCC1 219–300 ; and pDEST17 (Invitrogen) for XRCC1 1–183 and XRCC1 301–415 .

Techniques: Binding Assay, Activity Assay, Electrophoretic Mobility Shift Assay

Journal: Scientific Reports

Article Title: Identification of an XRCC1 DNA binding activity essential for retention at sites of DNA damage

doi: 10.1038/s41598-019-39543-1

Figure Lengend Snippet: Comparison of XRCC1-CDB binding affinities for different DNA substrates. ( a ) Schematic of DNA substrates analyzed for XRCC1-CDB interaction. All substrates contained a 3′-hydroxyl group at the site of damage. Substrates that contained a 5′-phosphate group at the damage site are labelled. ( b ) Comparison of binding curves of XRCC1-CDB with each substrate showed only minor differences in affinity.

Article Snippet: Primers used in PCR reactions for vector construction are listed in Supplementary Fig. . PCR products were first moved into a pDONR201 entry vector (Invitrogen) and subsequently recombined into destination vectors, pDEST15 (Invitrogen) for full length XRCC1; pDEST544 (Addgene 11519) for XRCC1 219–415 and XRCC1 219–300 ; and pDEST17 (Invitrogen) for XRCC1 1–183 and XRCC1 301–415 .

Techniques: Binding Assay

Journal: Scientific Reports

Article Title: Identification of an XRCC1 DNA binding activity essential for retention at sites of DNA damage

doi: 10.1038/s41598-019-39543-1

Figure Lengend Snippet: Small angle X-ray scattering of the XRCC1-CDB DNA-bound complex. Pair distribution curves (left) and resulting molecular envelopes (right) of ( a ) 39 bp DNA and ( b ) XRCC1-CDB with the estimated dimensions given in angstroms. An ab initio model for XRCC1-CDB was generated with DAMMIF (grey model) with subsequent BUNCH modelling (purple) to populate atoms not present in the BRCT1 domain determined by NMR (PDB 2D8M). ( c ) The pair distribution function for the XRCC1-CDB DNA-bound complex is shown in the left side of the panel. A corresponding MONSA generated model is shown in stereo. Purple spheres correspond to XRCC1-CDB while the yellow spheres correspond to 39 bp DNA.

Article Snippet: Primers used in PCR reactions for vector construction are listed in Supplementary Fig. . PCR products were first moved into a pDONR201 entry vector (Invitrogen) and subsequently recombined into destination vectors, pDEST15 (Invitrogen) for full length XRCC1; pDEST544 (Addgene 11519) for XRCC1 219–415 and XRCC1 219–300 ; and pDEST17 (Invitrogen) for XRCC1 1–183 and XRCC1 301–415 .

Techniques: Generated

Journal: Scientific Reports

Article Title: Identification of an XRCC1 DNA binding activity essential for retention at sites of DNA damage

doi: 10.1038/s41598-019-39543-1

Figure Lengend Snippet: Comparison of DNA Binding affinity for XRCC1-CDB mutants. ( a ) Sequence of the XRCC1 N-terminal linker. Positively charged residues that were targeted for alanine substitution are highlighted in bold. ( b ) Binding curves generated from EMSA analysis for each alanine substituted mutant (right) and the corresponding K d values (left). ( c ) Sequence alignment of XRCC1 from human, hamster, frog and Arabidopsis (plant). Conserved positively charged residues, green; glycine and proline, cyan; negatively charged residues, pink; and hydrophobic residues, yellow. ( d ) A comparison of DNA binding levels for mutant and wild type XRCC1 at 2 µM protein concentration. Each experiment was repeated three times. Mutant P1/3 had no measurable DNA binding activity at this concentration.

Article Snippet: Primers used in PCR reactions for vector construction are listed in Supplementary Fig. . PCR products were first moved into a pDONR201 entry vector (Invitrogen) and subsequently recombined into destination vectors, pDEST15 (Invitrogen) for full length XRCC1; pDEST544 (Addgene 11519) for XRCC1 219–415 and XRCC1 219–300 ; and pDEST17 (Invitrogen) for XRCC1 1–183 and XRCC1 301–415 .

Techniques: Binding Assay, Sequencing, Generated, Mutagenesis, Protein Concentration, Activity Assay, Concentration Assay

Journal: Scientific Reports

Article Title: Identification of an XRCC1 DNA binding activity essential for retention at sites of DNA damage

doi: 10.1038/s41598-019-39543-1

Figure Lengend Snippet: Comparison of XRCC1 foci formation following 10 mM H2O2 treatment. ( a ) Cells expressing wild type XRCC1 (left) or P1/3 variant (right). DNA, stained with DAPI (blue colour); XRCC1 fused with YFP (green). One single confocal image taken at the center of the nucleus is presented. ( b ) 3D-stacks were acquired and visualized with Volume Viewer. The colour code reflects the position of the foci in the 3D space. ( c ) The foci number were counted with the ImageJ plugin 3D Object Counter. Results for 7 representative cells expressing each of the XRCC1 variants are displayed in the scatter dot plot. Graph representation and Mann-Whitney statistical test were performed with GraphPad Prism version 7, 02. Scale bars, 5 µm.

Article Snippet: Primers used in PCR reactions for vector construction are listed in Supplementary Fig. . PCR products were first moved into a pDONR201 entry vector (Invitrogen) and subsequently recombined into destination vectors, pDEST15 (Invitrogen) for full length XRCC1; pDEST544 (Addgene 11519) for XRCC1 219–415 and XRCC1 219–300 ; and pDEST17 (Invitrogen) for XRCC1 1–183 and XRCC1 301–415 .

Techniques: Expressing, Variant Assay, Staining, MANN-WHITNEY

Journal: Scientific Reports

Article Title: Identification of an XRCC1 DNA binding activity essential for retention at sites of DNA damage

doi: 10.1038/s41598-019-39543-1

Figure Lengend Snippet: Effect of P1/3 mutation on DNA repair of single strand breaks. Relative repair efficiency of single strand breaks compared for CHO cells containing an XRCC1 knock out and the same cells complemented with either WT or P1/3 mutant XRCC1. The average tail moments are shown for each cell line after 30 min of recovery following initial damage with 10 mM hydrogen peroxide. Representative images from comet assays, which were used for average tail moment calculation, are provided in the lower panels.

Article Snippet: Primers used in PCR reactions for vector construction are listed in Supplementary Fig. . PCR products were first moved into a pDONR201 entry vector (Invitrogen) and subsequently recombined into destination vectors, pDEST15 (Invitrogen) for full length XRCC1; pDEST544 (Addgene 11519) for XRCC1 219–415 and XRCC1 219–300 ; and pDEST17 (Invitrogen) for XRCC1 1–183 and XRCC1 301–415 .

Techniques: Mutagenesis, Knock-Out