Detecting Pol II Phosphorylation and IL-1β Expression

To detect Pol II phosphorylation under drug treatment, total proteins from GK cells were extracted with RIPA buffer (Millipore) and resolved on 6% SDS page. After transferring to PVDF membrane, Pol II was probed with antibody against total Pol II (clone N20, Santa Cruz, Dallas, TX), Pol II CTD P-ser2 phosphorylation clone H5 (abcam, Cambridge, UK), and Pol II CTD P-ser5 phosphorylation (abcam). For detecting IL-1B expression, 100 μl culture medium from each time points and treatments were collected and the antigen was detected with a rabbit anti-grouper IL-1β antibody prepared in-house [15 (link)]. The results of the ELISA assay were acquired using a Varioskan Flash spectral scanning multimode plate reader (ThermoScientific).

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Lee F.F., Hui C.F., Chang T.H, & Chiou P.P. (2016). Alternative Splicing of Toll-Like Receptor 9 Transcript in Teleost Fish Grouper Is Regulated by NF-κB Signaling via Phosphorylation of the C-Terminal Domain of the RPB1 Subunit of RNA Polymerase II. PLoS ONE, 11(9), e0163415.

Publication 2016

RIPA buffer Varioskan Flash spectral scanning multimode plate reader

Anti antibody Antibody Antigen Assay Buffer Cells Clone Culture medium Drug Elisa Il 1β Membrane Phosphorylation Pol ii Proteins Pvdf Rabbit Ripa Sds page

Corresponding Organization : Genomics Research Center, Academia Sinica

Other organizations : Institute of Cellular and Organismic Biology, Academia Sinica

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Variable analysis

independent variables

Drug treatment

dependent variables

Pol II phosphorylation (Pol II CTD P-ser2, Pol II CTD P-ser5)
IL-1B expression

control variables

Total Pol II (clone N20, Santa Cruz, Dallas, TX)

controls

Positive control: Not specified
Negative control: Not specified

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