Generation of Inducible TRIP4 Variant Expressing Cell Lines

Stably transfected Flp-In™ T-REx™ 293 cell lines (Thermo Fisher Scientific R78007) for the tetracycline-inducible expression of TRIP4 variants with N-terminal 2xFlag-His₆ or C-terminal His₆-2xFlag tags were generated according to the manufacturer’s guidelines^{10 (link)}. Transfection of the parental cell line was done using X-tremeGENE HP DNA Transfection Reagent (Sigma-Aldrich). After hygromycin-based selection of cells that had genomically integrated the expression cassette, tetracycline-induced expression of the tagged proteins was confirmed by western blotting using a monoclonal α-Flag-M2 antibody (Sigma-Aldrich F3165; 1:7500). For expression of HA-tagged TRIP4 variants, the pHAGE-HA-trip4 vectors encoding HA-tagged TRIP4^wt, TRIP4^ΔZnF, TRIP4^{L174A-L180A-I190A} or TRIP4^C171A-C184A, were transfected into 293T cells (ATCC CRL-3216) using Transit293 transfection reagent (Mirus Bio).

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Jia J., Hilal T., Bohnsack K.E., Chernev A., Tsao N., Bethmann J., Arumugam A., Parmely L., Holton N., Loll B., Mosammaparast N., Bohnsack M.T., Urlaub H, & Wahl M.C. (2023). Extended DNA threading through a dual-engine motor module of the activating signal co-integrator 1 complex. Nature Communications, 14, 1886.

Publication 2023

Flp-In™ T-REx™ 293 cell lines X-tremeGENE HP DNA Transfection Reagent α-Flag-M2 antibody 293T cells Transit293 transfection reagent

293t cells Cell line Cells His6 tags Hygromycin Monoclonal antibody Parental Phage Proteins T cell Tetracycline Transfection Vectors

Corresponding Organization : Helmholtz-Zentrum Berlin für Materialien und Energie

Other organizations : Harvard University, Boston University, Universitätsmedizin Göttingen, Max Planck Institute for the Study of Religious and Ethnic Diversity, University of Göttingen

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Variable analysis

independent variables

Tetracycline-inducible expression of TRIP4 variants
N-terminal 2xFlag-His6 or C-terminal His6-2xFlag tags on TRIP4 variants
HA-tagged TRIP4 variants (TRIP4wt, TRIP4ΔZnF, TRIP4L174A-L180A-I190A, TRIP4C171A-C184A)

dependent variables

Tetracycline-induced expression of the tagged proteins
Protein expression levels of HA-tagged TRIP4 variants

control variables

Parental Flp-In™ T-REx™ 293 cell line
293T cell line

controls

Positive control: Monoclonal α-Flag-M2 antibody used to confirm tetracycline-induced expression of the tagged proteins
Negative control: Not explicitly mentioned

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