|
|
|
| The Analysis and Preparation of the Stress Associated Phosphorylation Antibodies Specific for the T103 Site of Tudor-SN Protein |
| GAO Xing-jie1, ZHANG Yi2, SU Chao1, FU Xue1, SHI Xue-bin3, YIN Jie1, HE Jin-yan3, WANG Xin-ting3, YAO Zhi3, YANG Jie1,3 |
1. Basic Medical College and Research Center, Tianjin Medical University, Tianjin 300070, China;
2. College of Pharmacy Tianjin Medical University, Tianjin 300070, China;
3. School of Basic Medical Sciences, Tianjin Medical University, Tianjin 300070, China |
|
|
|
Abstract Objective: To prepare and analyze the rabbit polyclonal stress associated phosphorylation antibodies specific for the T103 (Thr103) site of Tudor-SN protein. Methods: The phosphorylated T103 (pT103) site-containing polypeptide was synthesized; Antiserums were collected from the New Zealand white rabbits immunized with the polypeptide four times and purified through AKTA protein purification system. The Western blotting analysis,immunofluorescence experiment and In-cell Western assay were performed using the purified anti-pT103 antibodies. Results: (1) The phosphorylated peptide "TIENKpTPQGRC" was synthesized and about 75 ml rabbit antiserums were collected (2.08 mg/ml); (2) The phosphorylation signal was detected and increased under oxidative stress conditions in Western blotting analysis; the granule signal was also observed in the cytoplasm of HeLa cells and co-localized with the endogenous Tudor-SN-labeled stress granule during stress. (3) The phosphorylation level of Tudor-SN at T103 site fluctuates during the oxidative stress and after the removal of stress. Conclusion: The rabbit polyclonal anti-pT103 antibodies specific for the T103 site of Tudor-SN were prepared successfully. It will help to study the role of Tudor-SN phosphorylation modification in cellular stress responses.
|
|
Received: 08 April 2014
Published: 25 June 2014
|
|
|
|
Cite this article:
GAO Xing-jie, ZHANG Yi, SU Chao, FU Xue, SHI Xue-bin, YIN Jie, HE Jin-yan, WANG Xin-ting, YAO Zhi, YANG Jie. The Analysis and Preparation of the Stress Associated Phosphorylation Antibodies Specific for the T103 Site of Tudor-SN Protein. China Biotechnology, 2014, 34(06): 55-60.
URL:
https://manu60.magtech.com.cn/biotech/10.13523/j.cb.20140608 OR https://manu60.magtech.com.cn/biotech/Y2014/V34/I06/55
|
|
|
|
[1] Valineva T, Yang J, Palovuor I R, et al. The transcriptional co-activator protein p100 recruits histone acetyl transferase activity to STAT6 and mediates interaction between the CREB-binding protein and STAT6. J Biol Chem, 2005, 280(15): 14989-14996.
[2] Gao X, Zhao X, Zhu Y, et al. Tudor staphylococcal nuclease (Tudor-SN) articipates in small ribonucleoprotein (snRNP) assembly viainteracting with symmetrically dimethylated Sm proteins. J Biol Chem, 2012, 287(22): 18130-18141.
[3] Sundstrm J F, Vaculova A, Smertenko A P, et al. Tudor staphylococcal nuclease is an evolutionarily conserved component of the programmed cell death degradome. Nat Cell Biol, 2009, 11(11): 1347-1354.
[4] Gao X, Ge L, Shao J, et al. Tudor-SN interacts with and co-localizes with G3BP in stress granules under stress conditions. FEBS Lett, 2010, 584(16):3525-3532.
[5] Buchan J R, Parker R. Eukaryotic stress granules: the ins and outs of translation. Mol Cell, 2009, 36(6): 932-941.
[6] Thomas M G, Loschi M, Desbats M A, et al. RNA granules: the good, the bad and the ugly. Cell Signal, 2011, 23(2): 324-334.
[7] Erickson S L, Lykke-Andersen J. Cytoplasmic mRNP granules at a glance. J Cell Sci, 2011, 124(Pt3): 293-297.
[8] Maroney P A, Yu Y, Fisher J, et al. Evidence that microRNAs are associated with translating messenger RNAs in human cells. Nat Struct Mol Biol, 2006, 13(12): 1102-1107.
[9] Ohn T, Anderson P. The role of posttranslational modifications in the assembly of stress granules. Wiley Interdiscip Rev RNA, 2010, 1(3): 486-493.
[10] Mokas S, Mills J R, Garreau C, et al. Uncoupling stress granule assembly and translation initiation inhibition. Mol Biol Cell, 2009, 20(11): 2673-2683.
[11] Tourriere H, Chebli K, Zekri L, et al. The RasGAP-associated endoribonuclease G3BP assembles stress granules. J Cell Biol, 2003, 160(6): 823-831.
[12] Weissbach R, Scadden A D. Tudor-SN and ADAR1 are components of cytoplasmic stress granules. RNA, 2012, 18(3): 462-471.
[13] Zhu L, Tatsuke T, Mon H, et al. Characterization of Tudor-sn-containing granules in the silkworm, Bombyx mori. Insect Biochem Mol Biol, 2013, 43(8): 664-674.
[14] Spector D L. SnapShot: Cellular Bodies Cell, 2006, 127(5):1071.
|
|
Viewed |
|
|
|
Full text
|
|
|
|
|
Abstract
|
|
|
|
|
Cited |
|
|
|
|
| |
Shared |
|
|
|
|
| |
Discussed |
|
|
|
|
