A20 mutant plasmids were created using QuickChange Site-Directed Mutagenesis kit (Agilent Technologies) to wt TNFAIP3 obtained from the human Orfeome collection (Horizon Discovery). TNFAIP3 constructs were further subcloned into N-terminal MAC-tag Gateway destination vector.24 (link) All generated constructs were confirmed with direct sequencing. The MAC-tagged expression constructs were transfected into Flp-In 293 T-REx cells (Invitrogen, Life Technologies) with FuGENE HD (Promega). The cells were grown according to manufacturer’s instructions under selection with Hygromycin B (Thermo Fisher Scientific) to create stable cell lines. Expression of stable transgenes was confirmed by western blotting using anti-hemagglutin (HA) primary (Biolegend, MMS-101R) and horseradish peroxidase-conjugated secondary antibody.
Protocol detail
Generating A20 Mutant Plasmids and Stable Cell Lines
A20 mutant plasmids were created using QuickChange Site-Directed Mutagenesis kit (Agilent Technologies) to wt TNFAIP3 obtained from the human Orfeome collection (Horizon Discovery). TNFAIP3 constructs were further subcloned into N-terminal MAC-tag Gateway destination vector.24 (link) All generated constructs were confirmed with direct sequencing. The MAC-tagged expression constructs were transfected into Flp-In 293 T-REx cells (Invitrogen, Life Technologies) with FuGENE HD (Promega). The cells were grown according to manufacturer’s instructions under selection with Hygromycin B (Thermo Fisher Scientific) to create stable cell lines. Expression of stable transgenes was confirmed by western blotting using anti-hemagglutin (HA) primary (Biolegend, MMS-101R) and horseradish peroxidase-conjugated secondary antibody.
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293 t cells
Antibody
Cell lines
Cells
Fugene
Horseradish peroxidase
Human
Hygromycin b
Plasmids
Promega
Site directed mutagenesis
Tnfaip3
Transgenes
Vector
Corresponding Organization : University of Helsinki
Other organizations : Helsinki University Hospital, University of Oulu, Oulu University Hospital, Institute for Molecular Medicine Finland, Lapland Central Hospital, Invalidisäätiö
Top 5 similar protocols
Variable analysis
independent variables
- A20 mutant plasmids
- Expression of stable transgenes
- Flp-In 293 T-REx cells
- Hygromycin B selection
- Positive control: Western blotting using anti-hemagglutin (HA) primary and horseradish peroxidase-conjugated secondary antibody
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