Journal: PLoS Genetics

Article Title: The Role of the Mammalian DNA End-processing Enzyme Polynucleotide Kinase 3’-Phosphatase in Spinocerebellar Ataxia Type 3 Pathogenesis

doi: 10.1371/journal.pgen.1004749

Figure Lengend Snippet: The spot annotated within the dashed box (absent in the control IP) was identified as human ATXN3 by subsequent MS/MS analysis .

Article Snippet: In situ Proximity Ligation Assay (PLA) between PNKP (anti-mouse Ab, a gift from Michael Weinfeld) and ATXN3 (anti-rabbit Ab, Proteintech) was carried out according to the protocol as described [ ] using a Duolink PLA kit (QLink Bioscience Cat# LNK 92101 K101, Uppsala, Sweden).

Techniques: Control, Tandem Mass Spectroscopy

Journal: PLoS Genetics

Article Title: The Role of the Mammalian DNA End-processing Enzyme Polynucleotide Kinase 3’-Phosphatase in Spinocerebellar Ataxia Type 3 Pathogenesis

doi: 10.1371/journal.pgen.1004749

Figure Lengend Snippet: HEK-293 cells were transfected with either PNKP siRNA or ATXN3 siRNA or control siRNA and the nuclear extracts (1 mg) prepared from those cells were IP’d with anti-PNKP Ab (BioBharati Life Science Pvt. Ltd, Kolkata, India; 2A ) or anti-ATXN3 Ab (Proteintech, 2B ) or with IgG as a control, and tested for the presence of PNKP- and ATXN3-associated proteins with Abs to the proteins shown on the right. (C) Detection of PNKP’s interaction with ATXN3 in SH-SY5Y cells by proximity ligation assays using a Duolink kit (Olink Bioscience, Uppsala, Sweden). Nuclei were counterstained with DAPI (blue). ATXN3-depleted (by siRNA) cells were used as a control to show the specificity of the interaction (middle panel). Right panel, Non-specific Ab (IgG) control (D) GST-PNKP pull-down of ATXN3 (WT and mutant) using purified GST-tagged full-length or three domains (FHA-, Kinase- and Phosphatase-domain) of PNKP, probed with anti-ATXN3. Bottom panel, Coomassie-stained gel of the corresponding PNKP domains, a second gel run in parallel.

Article Snippet: In situ Proximity Ligation Assay (PLA) between PNKP (anti-mouse Ab, a gift from Michael Weinfeld) and ATXN3 (anti-rabbit Ab, Proteintech) was carried out according to the protocol as described [ ] using a Duolink PLA kit (QLink Bioscience Cat# LNK 92101 K101, Uppsala, Sweden).

Techniques: Transfection, Control, Ligation, Mutagenesis, Purification, Staining

Journal: PLoS Genetics

Article Title: The Role of the Mammalian DNA End-processing Enzyme Polynucleotide Kinase 3’-Phosphatase in Spinocerebellar Ataxia Type 3 Pathogenesis

doi: 10.1371/journal.pgen.1004749

Figure Lengend Snippet: (A) A 32 P-labeled 3’-phosphate-containing oligo substrate (5 pmol) was incubated at 37°C for 10 min in buffer A (25 mM Tris-HCl, pH 7.5, 100 mM NaCl, 5 mM MgCl 2 , 1 mM DTT, 10% glycerol and 0.1 μg/μl acetylated BSA) with PNKP alone (50 fmol, ln 2) or ATXN3-Q29 alone (400 fmol, ln 8) or PNKP plus increasing amounts of Q29 (25, 50, 100, 200 and 400 fmol; lns 3–7). Ln 1, substrate only. Generation of the 32 P-labelled 3’P-containing oligo substrate was described previously . (B) Effect of mutant ATXN3 (Q72) on PNKP’s 3’-phosphatase activity. 32 P-labelled 3’-phosphate-containing oligo substrate (2.5 pmol) was incubated with PNKP at 37°C for 20 min alone (50 fmol, ln 2), ATXN3-Q72 alone (200 fmol, ln 3), PNKP (50 fmol) plus increasing amounts of Q72 (50, 100 and 200 fmol, lns 4–6). Ln 1, no protein, substrate only. Quantitation of the products (released phosphate) is represented in the histogram (lower panel), with the activity of PNKP alone arbitrarily set as 1 (n = 3, ** = P< 0.01).

Article Snippet: In situ Proximity Ligation Assay (PLA) between PNKP (anti-mouse Ab, a gift from Michael Weinfeld) and ATXN3 (anti-rabbit Ab, Proteintech) was carried out according to the protocol as described [ ] using a Duolink PLA kit (QLink Bioscience Cat# LNK 92101 K101, Uppsala, Sweden).

Techniques: Labeling, Incubation, Mutagenesis, Activity Assay, Quantitation Assay

Journal: PLoS Genetics

Article Title: The Role of the Mammalian DNA End-processing Enzyme Polynucleotide Kinase 3’-Phosphatase in Spinocerebellar Ataxia Type 3 Pathogenesis

doi: 10.1371/journal.pgen.1004749

Figure Lengend Snippet: (A) Western blot analysis showing the level of expression of ATXN3 in SH-SY5Y cells stably expressing control vs. ATXN3-shRNA. Lamin B was used as a loading control. (B) A representative gel (n = 3) showing the 3’-phosphatase activity in the nuclear extract (200 ng) of control (ln 3) and ATXN3-depleted cells (ln 4). Lns 5–8, increasing amounts (10, 25, 50 and 100 fmol) of purified ATXN3 (WT) were added back to ATXN3-depleted nuclear extract. Quantitation of the products is represented in the histogram (n = 3, ** = P< 0.01).

Article Snippet: In situ Proximity Ligation Assay (PLA) between PNKP (anti-mouse Ab, a gift from Michael Weinfeld) and ATXN3 (anti-rabbit Ab, Proteintech) was carried out according to the protocol as described [ ] using a Duolink PLA kit (QLink Bioscience Cat# LNK 92101 K101, Uppsala, Sweden).

Techniques: Western Blot, Expressing, Stable Transfection, Control, shRNA, Activity Assay, Purification, Quantitation Assay

Journal: PLoS Genetics

Article Title: The Role of the Mammalian DNA End-processing Enzyme Polynucleotide Kinase 3’-Phosphatase in Spinocerebellar Ataxia Type 3 Pathogenesis

doi: 10.1371/journal.pgen.1004749

Figure Lengend Snippet: Long amplicon qPCR (LA-QPCR) was used to evaluate genomic DNA SB levels in control vs. ATXN3-depleted HEK-293 cells. Representative gel showing PCR-amplified fragments of the HPRT (left panel) and POLB (right panel) genes. Amplification of each large fragment (upper panels) was normalized to that of a small fragment of the corresponding gene (bottom panels) and the data were expressed as lesion frequency/10 Kb DNA as described previously . Histograms represent the DNA damage quantitation for control vs ATXN3-depleted cells (n = 3, ** = P< 0.01). Error bars indicate standard error of means.

Article Snippet: In situ Proximity Ligation Assay (PLA) between PNKP (anti-mouse Ab, a gift from Michael Weinfeld) and ATXN3 (anti-rabbit Ab, Proteintech) was carried out according to the protocol as described [ ] using a Duolink PLA kit (QLink Bioscience Cat# LNK 92101 K101, Uppsala, Sweden).

Techniques: Amplification, Control, Quantitation Assay

Journal: PLoS Genetics

Article Title: The Role of the Mammalian DNA End-processing Enzyme Polynucleotide Kinase 3’-Phosphatase in Spinocerebellar Ataxia Type 3 Pathogenesis

doi: 10.1371/journal.pgen.1004749

Figure Lengend Snippet: (A) Upper panel, 32 P-labelled 3’-phosphate-containing oligo substrate (2.5 pmol) was incubated with the NE (250 ng) isolated from rat mesenchymal (CSM14.1) cells conditionally expressing vector alone (lane 1), WT (Q-23, lane 2) or mutant ATXN3 (Q-70, lane 3). Ln 4, no protein. S: substrate, P: product (released 32 P i ). Middle panel: Western analysis of the corresponding NE (10μg) showing PNKP expression. GAPDH (Ab from Genetex Inc.) was used as loading control. Bottom panel: quantitation of the products (P) is represented in a histogram. (n = 3, ** = P< 0.01) (B) Comet assays showing the accumulation of DNA SBs in mutant (Q-70) ATXN3 cells. (C) Comparative (WT vs. SCA3 mice) 3’-phosphatase activity in the NEs from four different brain regions. 7.5 pmol of 3’-P-containing substrate was incubated at 37°C for 10 min with WT or transgenic mouse brain NE (200 ng; PN-Pontine Nuclei, SN-Substantia Nigra, DCN-Deep Cerebellar Nuclei, and HP-Hippocampus). Quantitation of the products is shown in the histogram with the age-matched WT arbitrarily set as 1 (n = 5; ** = P< 0.01).

Article Snippet: In situ Proximity Ligation Assay (PLA) between PNKP (anti-mouse Ab, a gift from Michael Weinfeld) and ATXN3 (anti-rabbit Ab, Proteintech) was carried out according to the protocol as described [ ] using a Duolink PLA kit (QLink Bioscience Cat# LNK 92101 K101, Uppsala, Sweden).

Techniques: Incubation, Isolation, Expressing, Plasmid Preparation, Mutagenesis, Western Blot, Control, Quantitation Assay, Activity Assay, Transgenic Assay