- References (4)
- Inventor Info
|Antigen/Gene or Protein Targets||Clathrin adaptor AP-2 (alpha-adaptin isoform C)|
|Disease Keywords||Protein transport; cell trafflicking machinery; hereditary spastic paraplegias|
The HeLa-Mitotrap-Ap2a2-FKBP cell line has been designed to provide new insights into the adaptor protein complex 2 (AP-2) and AP-2’s binding partners and its function in cell trafflicking.
This type of approach, a “knocksideways,” inactivates proteins by using a small molecule to trap them onto mitochondria. It is typically 3 to 4 orders of magnitude faster than a knockdown.
The HeLa-Mitotrap-Ap2a2-FKBP cell line was generated by transfecting the HeLa-Mitotrap cell line with a second construct expressing FKBP-tagged subunits of the adaptor protein (AP2). An FKBP domain is a cellular chaperone protein and prolyl isomerase which is a target for rapamycin binding and dimerizes with FRB-containing protein. Upon treatment with rapamycin the target protein containing an FKBP domain, (e.g, AP2), will dimerize with FRB domains and the resulting protein complexes will be sequestered to the mitochondria within minutes of treatment rendering the cells ready for immediate assay.
|Production Details||HeLa-Mitotrap expresses a mitochondrial trapping construct with an FRB domain and is maintained in hygromycin-containing medium. The other three cell lines also express Mitotrap, and in addition they express a protein of interest with an FKBP domain attached, which can be trapped onto mitochondrial when rapamycin is added. The three proteins of interest are Ap1g1 or gamma-adaptin; Ap2a2 or alpha-adaptin isoform C; and GGA2.|
|Conditional Description||Rapamicin-induced knocksideways|
|Research Area||Cell Signaling & Signal Transduction, Cellular Transport|
|Recommended Growing Conditions||HeLa cells were cultured in Dulbecco’s Modified Eagle Medium supplemented with 10% fetal bovine serum (FBS) and 100 U/ml penicillin/streptomycin at 37°C and 5% CO2. Cells were transfected using GeneJuice. Rapamycin was used at 200 nM, vehicle was ethanol (0.1%). MLN8237 was used at 1 µM, vehicle was DMSO (0.01%).|
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References: 4 entries
References: 4 entries