Subcellular Fractionation and Immunoblotting

Cells were harvested and suspended in buffer A (10 mM HEPES (pH 7.4), 10 mM KCl, 1 mM dithiothreitol, 0.6% NP-40) (TransGen Biotech, Shanghai, China) [59 (link)]. We collected the supernatant as the cytoplasmic fraction after centrifugation. We suspended the remaining pellet in nuclear extraction buffer (20 mM HEPES (pH 7.4), 150 mM NaCl, 1 mM dithiothreitol) (TransGen Biotech, Manchester, United Kingdom), and collected the supernatant as the nuclear fraction after centrifugation [59 (link)]. We used equal amounts of cytoplasmic and nuclear extracts for immunoblotting of STAT1, Nmi, actin, and histone H1 (Proteintech, Manchester, United Kingdom).

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Feng L., Sheng J., Vu G.P., Liu Y., Foo C., Wu S., Trang P., Paliza-Carre M., Ran Y., Yang X., Sun X., Deng Z., Zhou T., Lu S., Li H, & Liu F. (2018). Human cytomegalovirus UL23 inhibits transcription of interferon-γ stimulated genes and blocks antiviral interferon-γ responses by interacting with human N-myc interactor protein. PLoS Pathogens, 14(1), e1006867.

Publication 2018

STAT1

Actin Buffer Cells Centrifugation Cytoplasmic Dithiothreitol Hepes Histone h1 Nacl Np 40 Stat1

Corresponding Organization : University of Southern California

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Variable analysis

independent variables

Buffer A (10 mM HEPES (pH 7.4), 10 mM KCl, 1 mM dithiothreitol, 0.6% NP-40)
Nuclear extraction buffer (20 mM HEPES (pH 7.4), 150 mM NaCl, 1 mM dithiothreitol)

dependent variables

Cytoplasmic fraction
Nuclear fraction
Immunoblotting of STAT1, Nmi, actin, and histone H1

control variables

Equal amounts of cytoplasmic and nuclear extracts used for immunoblotting

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