Binding Assays Using Recombinant SH2 Domains: Far-Western, Pull-Down, and Fluorescence Polarization

Machida, Kazuya; Liu, Bernard A.

doi:10.1007/978-1-4939-6762-9_17

Cited by 4 publications

(2 citation statements)

References 14 publications

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“…GST pull-down assay was done as previously reported [21]. Briefly, cells were gathered, rinsed using PBS, re-suspended in binding buffer and sonicated.…”

Section: Glutathione Sepharose-transferase (Gst) Pulldown Assaymentioning

confidence: 99%

Sirt1 modulates H3 phosphorylation and facilitates osteosarcoma cell autophagy

Ying

Zhang

et al. 2019

Artificial Cells, Nanomedicine, and Biotechnology

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Abnormal histone modifications have been recognized as an important contributing factor to the initiation and progression of osteosarcoma. Sirtuin 1 (Sirt1) up-regulation has been discovered in osteosarcoma cells. This study tested the influence of Sirt1 on histone H3 phosphorylation at threonine 3 (H3 T3ph ) in osteosarcoma cells, along with Sirt1-H3 T3ph axis on osteosarcoma cell autophagy. Plasmids or si-RNAs transfection was carried out to alter Sirt1 or H3 T3ph expressions. Co-immunoprecipitation analysis and GST pull-down assay were done to probe the relationship between Sirt1 and H3 T3ph . Phosphoryltransferase activity of Sirt1 was tested by in vitro kinase activity assay. Cell autophagy was measured by a number of autophagosome, conversion of LC3-I to LC3-II, degradation of long-lived protein and ATG protein expressions. We found that Sirt1 directly interacted with H3 and phosphorylate H3 T3 at threonine 3 in osteosarcoma cells. Moreover, Sirt1 facilitated osteosarcoma cell autophagy under starvation condition. H3 T3ph took part in the Sirt1-facilitated osteosarcoma cell autophagy under starvation condition. Besides, Sirt1-H3 T3ph axis facilitated osteosarcoma cell autophagy might be achieved through transcriptional activation of ATG genes. Sirt1 promoted osteosarcoma initiation and progression might be via phosphorylate H3 T3 and then facilitate osteosarcoma cell autophagy through activating ATG genes transcription under starvation condition. ARTICLE HISTORY

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“…GST pull-down assay was done as previously reported [21]. Briefly, cells were gathered, rinsed using PBS, re-suspended in binding buffer and sonicated.…”

Section: Glutathione Sepharose-transferase (Gst) Pulldown Assaymentioning

confidence: 99%

Sirt1 modulates H3 phosphorylation and facilitates osteosarcoma cell autophagy

Ying

Zhang

et al. 2019

Artificial Cells, Nanomedicine, and Biotechnology

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“…GST pull-down assay was done as earlier described [21]. Briefly, GST-tagged JHDM1A was expressed in Escherichia coli was mixed with GS-4B beads (GE healthcare, Beijing, China) and spun down at 4 C. Followed by rinsing, the beads were reacted with in vitro-transcribed/translated HA-tagged p300.…”

Section: Glutathione S-transferase (Gst) Pull Down Assaymentioning

confidence: 99%

p300 Acetylates JHDM1A to inhibit osteosarcoma carcinogenesis

Wang

Sun

Zhang

et al. 2019

Artificial Cells, Nanomedicine, and Biotechnology

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JHDM1A participates in cancer development via demethylate dimethyl histone H3 lysine 36 (H3K36me2). p300 is an intrinsic acetyltransferase. This study explored the acetyltransferase activity of p300 on JHDM1A and analyzed the JHDM1A acetylation on H3K36me2 demethylation in osteosarcoma. Co-immunoprecipitation (CoIP) and immunoblotting assay found that p300 directly acetylated JHDM1A at K409 residue in osteosarcoma MG-63 and HOS cells. Nucleosomes and mononucleosomes were prepared and found that acetylation of JHDMIA disrupted its association with nucleosomes and thereby impaired its capability to induce H3K36me2 demethylation. Moreover, chromatin immunoprecipitation (ChIP) assay discovered that the input levels of H3K36me2 in the promoter regions of p21 and puma were increased after acetylation of JHDM1A, which raised the p21 and puma mRNA levels in the cells. Finally, the analysis of JHDM1A acetylation on osteosarcoma cell proliferation and invasion, along with tumor growth pointed out that acetylation of JHDMIA inhibited the proliferation and invasion of osteosarcoma HOS cells, as well as suppressed the tumor growth of osteosarcoma. In conclusion, the outcomes of our research verified that p300 could directly acetylate JHDM1A at K409 site, which reduces the demethylation of H3K36me2, enhanced the transcription of p21 and puma, and thereby inhibited the growth and metastasis of osteosarcoma.

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