V5-APEX2 was PCR-amplified from the pcDNA5-Mito-V5-APEX2 plasmid, which was kindly provided by Dr. Hyun-Woo Rhee (Seoul National University). V5-APEX2 alone or IPMK-V5-APEX2 were cloned into the pcDNA5/FRT/TO plasmid (Invitrogen). Flp-In T-REx–293 (Invitrogen) cells were seeded in six-well culture plates to 70% confluency, and then co-transfected with 0.25 µg of pcDNA5/FRT/TO and 2.25 µg of pOG44 Flp recombinase expression plasmid (Invitrogen) using Lipofectamine LTX with Plus Reagent (Invitrogen). After 48 hr, the cells were transferred to 90 mm culture dishes for negative selection with 50 µg/ml Hygromycin B (Gibco) until all non-transfected cells were dead. Surviving cells were seeded at low confluency to generate cellular clones on culture plates, after which each clone was individually screened for APEX2 construct expression with or without doxycycline (Sigma Aldrich) to search for optimal cell populations with minimal uncontrolled APEX2 expression and maximal APEX2 expression under stimulation. Selected clones were then expanded and stored in liquid nitrogen for downstream experiments.
Pog44 flp recombinase expression plasmid
Manufactured by Thermo Fisher Scientific
The POG44 (Flp recombinase expression plasmid) is a lab equipment product that expresses the Flp recombinase enzyme. The Flp recombinase is a site-specific DNA recombinase derived from the 2-micron plasmid of Saccharomyces cerevisiae. The core function of this product is to facilitate the recombination of DNA sequences flanked by Flp recognition target (FRT) sites.
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Lab products found in correlation
Flp in t rex 293 cell line, by Thermo Fisher Scientific (2 mentions)
Hygromycin b, by Thermo Fisher Scientific (1 mentions)
Doxycycline, by Merck Group (1 mentions)
Pcdna5 frt to plasmid, by Thermo Fisher Scientific (1 mentions)
Flp in t rex 293, by Thermo Fisher Scientific (1 mentions)
Lipofectamine ltx with plus reagent, by Thermo Fisher Scientific (1 mentions)
3 protocols using pog44 flp recombinase expression plasmid
1
Inducible APEX2 Fusion Protein Expression
2
Generation of Stable Cell Lines Expressing FBXL2 Variants
3
Generation of Stable Cell Lines Expressing FBXL2 Variants
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FLAG-tagged FBXL2 wild-type, FBXL2(C420S) and FBXL2(C417S/C419S) inserts were obtained by restriction digestion from their respective pcDNA3 constructs and subcloned into pcDNA™5/FRT/TO (Invitrogen) by using HindIII and NotI (Invitrogen) restriction sites compatible between the two vectors. Individual constructs were co-transfected with pOG44 (Flp recombinase expression plasmid, Invitrogen) into the Flp-In™ T-Rex™-293 cell line (Invitrogen) containing a single integrated FRT (FLP Recombination Target) site and stably expressing the Tet repressor. FRT site present in the vector is required for its Flp recombinase mediated integration into the Flp-In™ T-Rex™ host cell line. Hygromycin (160 μg/mL) was used for the selection of positive cells. Doxycycline (2 μg/mL) was added to the medium for inducing gene expression.
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