Quantitative Analysis of Nuclear Import of Stress-Related Transcription Factors

Nuclear extracts were prepared from frozen livers as previously described [19 (link)]. Briefly, liver pieces were disrupted in a Dounce hand homogenizer on ice without NP-40 and nuclei pelleted at 4000 x g for 1 minute before extract preparation. Extracts from 5 mice in each group (sham-infected, CS-infected + vehicle and CS-infected + cSN50.1) were analyzed for nuclear import of SRTFs by quantitative immunoblotting using polyclonal goat anti-cFos (Santa Cruz), monoclonal mouse anti- Y701phophorylated STAT1 (Becton-Dickinson) and polyclonal rabbit anti-NF-κBp50 and anti-NF-κB p65 (Abcam) on a Licor Odyssey Infrared Imaging System. Mouse monoclonal anti-TATA binding protein (TBP, Abcam) was used to measure TBP as a nuclear loading control for normalization.

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Veach R.A., Liu Y., Zienkiewicz J., Wylezinski L.S., Boyd K.L., Wynn J.L, & Hawiger J. (2017). Survival, bacterial clearance and thrombocytopenia are improved in polymicrobial sepsis by targeting nuclear transport shuttles. PLoS ONE, 12(6), e0179468.

Publication 2017

Mouse monoclonal anti-TATA binding protein (TBP,

Frozen Goat Liver Mouse Nf κb p65 Np 40 Nuclear import Nuclei Rabbit Stat1

Corresponding Organization : University of Florida

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Variable analysis

independent variables

Treatment group (sham-infected, CS-infected + vehicle, CS-infected + cSN50.1)

dependent variables

Nuclear import of SRTFs (cFos, Y701phosphorylated STAT1, NF-κBp50, NF-κB p65)

control variables

TATA binding protein (TBP) as a nuclear loading control

controls

Positive control: Not specified
Negative control: Sham-infected group

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