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Objective To construct and identify the recombinant plasmid pmCherry-C1-Argonaute1 and to provide the basis for further research on the biological role of Argonaute1 protein in the field of cell stress and its related diseases. Methods The total RNA was extracted from Hela cells and c DNA containing Argonaute1 was inverse transcribed. Argonaute1 fragment containing EcoRI and Bam HI site was amplified by PCR from purified tamplate from He La cell line and inserted into pmCherry-C1 fluorescent expressing vector. The recombinant pmCherry-C1-Argonaute1 plasmid was transfected into He La cells and the expression of red fluorescent fusion proteins was examined by Western blotting and the co-localization in stress condition of Argonaute1 and G3BP( biomarker in stress granules) and DCP1α ( biomarker in processing bodies) was detected by fluorescence microscope. Results The recombinant plasmid was sequenced correctly which was identified by EcoRI,Bam HI,the restriction single / double enzyme digestion and gene sequencing. The red fluorescent fusion protein was also detected in transfected He La cell by Western blotting. The result of three-color immunofluorescence co-localization indicated that Cherry-red labeled Argonaute1 was co-localized with G3 BP and DCP1α in cells under stress condition. Conclusions The recombinant eukaryotic plasmid of pmCherry-C1-Argonaute1 is constructed successfully and expresses Cherry-red labeled Argonaute1 fusion protein effectively. These results will help to study the biological role of Argonaute1 in cell stress and related diseases. [ABSTRACT FROM AUTHOR]

Objective To construct eukaryotic Flag (DYKDDDDK) expressing recombinant plasmids, pCMV-N-Flag-SND1-No1/2, which contain the coding sequence of human SND1-No1 (from 1st AUG)or SND1-No2 (from 2nd AUG), and perform the cellular localization analysis of Flag-tagged SND1-No1/2 under stress condition to study the function of the two AUG in the SND1 containing stress granules formation. Methods The gene fragments of SND1-No1/2 were amplified by PCR from the whole SND1 transcript and inserted into pCMV-N-Flag expressing vector through BamHI/EcoRI double enzyme digestion and T4 DNA Ligase connection. The recombinant pCMV-N-Flag-SND1-No1/2 plasmids were transfected into HeLa cells and the expression of Flag-SND1-No1/2 fusion proteins was examined by Western blotting assay. Immunofluo-rescence assay was performed to detect the co-localization of Flag-SND1-No1/2 with endogenous SND1 granule. Results The pCMV-N-Flag-SND1-No1/2 were sequenced and digested correctly by restriction single/double enzyme. The Flag-tagged SND1-No1/2 fusion proteins were also detected in transfected HeLa cell by Western blotting assay. Both of them showed the co- localization with endogenous SND1 granule. Conclusion The recombinant eukaryotic plasmids of pCMV-N-Flag-SND1-No1/2 were constructed successfully and expressed effectively. The depletion of 1st AUG failed to affect the formation of SND1 containing stress granules. [ABSTRACT FROM AUTHOR]

Objective To construct eukaryotic enhanced green fluorescent protein (EGFP) expressing recombinant plasmid, pEGFP-C1-hnRNP A1, which contains coding sequence of human hnRNP A1 (heterogeneous nuclear ribonucleo protein A1), and to perform cellular localization analysis of EGFP tagged hnRNP A1 under stress. Methods Total RNA was isolated from HeLa cell used for synthesis of first-strand cDNAs using reverse primers that are specific for the3'-un-translated region of hnRNP A1. hnRNP A1 gene fragments were then amplified by touch-down PCR from those cDNAs and inserted into pEGFP-C1 fluorescent bearing vector through EcoR I/BamH II double enzyme digestion and T4 DNA Ligase connection. The recombinant pEGFP-C1-hnRNP A1 plasmid was transfected into HeLa cells and green fluorescent tagged fusion proteins was examined by Western blot and confocal fluorescence microscopy. Co-localization of EGFP-hnRNP A1 with poly (A)+ mRNA (the marker of the stress granules), or DCP1a (the marker of processome) were detected by RNA fluores cence in situ hybridization and immunofluorescence. Results The pEGFP-C1-hnRNP A1 was sequenced and digested correctly by restriction single/double enzyme. The green fluorescent fusion protein was also detected in transfected HeLa cell by Western blot and confocal fluorescence microscopy. EGFP-hnRNP A1 co-localizes with poly(A)+ mRNA, but not DCP1a. Conclusion Recombinant eukaryotic plasmid of pEGFP-C1-hnRNP A1 was constructed successfully and expressed effectively. EGFP tagged hnRNP A1 takes part in forming stress granules. [ABSTRACT FROM AUTHOR]