Name: Identification of PTB ΔNLS-GFP interacting proteins precipitated by magnetic beads against GFP
Keywords: Ptb

Dataset Information

Identification of PTB ΔNLS-GFP interacting proteins precipitated by magnetic beads against GFP

ABSTRACT: The purpose of the study was to determine proteins that specially interact cytoplasmic PTB. PTB is an RNA binding protein that shuttles between the nucleus and cytoplasm. To identified proteins interacting with cytoplasmic PTB, nucleus localization signal was deleted (PTB ΔNLS-GFP) and overexpressed followed by IP and MS.

INSTRUMENT(S): Q Exactive HF

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Epithelial Cell

DISEASE(S): Disease Free

SUBMITTER: Fuxing Gong

LAB HEAD: Fuxing Gong

PROVIDER: PXD026837 | Pride | 2022-02-17

REPOSITORIES: Pride

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Publications

De-dimerization of PTB is catalyzed by PDI and is involved in the regulation of p53 translation.

Gong Fu-Xing FX Zhan Guoqin G Han Rong R Yang Zhigang Z Fu Xin X Xiao Ran R

Nucleic acids research 20210901 16

Polypyrimidine tract-binding protein (PTB) is an RNA binding protein existing both as dimer and monomer and shuttling between nucleus and cytoplasm. However, the regulation of PTB dimerization and the relationship between their functions and subcellular localization are unknown. Here we find that PTB presents as dimer and monomer in nucleus and cytoplasm respectively, and a disulfide bond involving Cysteine 23 is critical for the dimerization of PTB. Additionally, protein disulfide isomerase (PD ...[more]

PMID: 34403458

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Project description:PTB is multifunctional RNA binding protein reported to be involved in splicing, 3' -end processing, stability and translational regulation. Sample preparation methods (Gama-Carvalho et al, 2006, submitted); Suspension HeLa cells were grown in DMEM, 10% FCS, Pen/Strep and split 1:2 the day before harvesting. Post-nuclear cytoplasmic extracts were obtained as described (Herbert and Hecht 1999), using 5x107 cells/ml of lysis buffer. mRNAs were isolated by immunoprecipitation from 200ïl of precleared HeLa cell extract, using the anti-U2AF65 MC3 mAb (Gama-Carvalho, Krauss et al. 1997) or the Bb7 anti-PTB mAb (ATCC) for 2h at 4ÂºC. Immune complexes were precipitated with a slurry with 50% of protein A/protein G agarose beads (Amersham), blocked with 100ïg/ïl of tRNA and RNAse free BSA (Ambion), by rotating for 1h at 4ÂºC and washed with lysis buffer. Complexes bound to the beads were eluted with TES buffer (10mM Tris, 0.5M EDTA, 0.5% SDS, pH 8.0) by heating at 65ÂºC for 10 minutes and Trizol extracted. Polyadenilated RNA in the cell extract (input) and in the immunoprecipitation sample was reverse transcribed, end-tagged and PCR amplified using the Super SMART PCR cDNA synthesis kit from Clontech, following manufacturer instructions. 1ïg of input or precipitated RNA was used per RT-PCR reaction to produce samples for microarray hybridization. For each set of samples (input and immunoprecipitated) a 100ïl test PCR reaction was performed according to manufacturer instructions and analyzed by agarose gel electrophoresis to select ideal conditions for amplification in the linear range. PCR reactions were pooled to obtain enough material for microarray hybridization. 15ïg of PCR amplified cDNA from input and immunoprecipitated samples were fragmented, end labeled with biotin and hybridized to Affymetrix GeneChip Human Genome U133 Plus 2.0 Arrays as described (Brodsky, Meyer et al. 2005), following standard Affymetrix hybridization protocols. Brodsky, A. S., C. A. Meyer, et al. (2005). "Genomic mapping of RNA polymerase II reveals sites of co-transcriptional regulation in human cells." Genome Biol 6(8): R64. Gama-Carvalho, M., R. D. Krauss, et al. (1997). "Targeting of U2AF65 to sites of active splicing in the nucleus." J Cell Biol 137(5): 975-87. Herbert, T. P. and N. B. Hecht (1999). "The mouse Y-box protein, MSY2, is associated with a kinase on non-polysomal mouse testicular mRNAs." Nucleic Acids Res 27(7): 1747-53. Experiment Overall Design: We have used microarray hybridization coupled to RNA immunoprecipitation from HeLa cell cytoplasmic extracts using anti-PTB antibodies to identify mRNA molecules associated with the PTB splicing factor

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	1.pdResult	Other
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	2.pdResult	Other
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	3.pdResult	Other