- Research Article
- Open Access
Establishing and functional characterization of an HEK-293 cell line expressing autofluorescently tagged β-actin (pEYFP-ACTIN) and the neurokinin type 1 receptor (NK1-R)
Cellular & Molecular Biology Letters volume 15, Article number: 55 (2010)
This study focused on establishing and making a comprehensive functional characterization of an HEK-293-transfected cell line that would coexpress the enhanced yellow fluorescent protein-actin (pEYFP-actin) construct and the neurokinin type 1 receptor (NK1-R), which is a member of the seven transmembrane (7TM) receptor family. In the initial selection procedure, the cloning ring technique was used alone, but failed to yield clones with homogenous pEYFP-actin expression. Flow cytometry sorting (FCS) was subsequently used to enrich the pEYFP-actin-expressing subpopulation of cells. The enzyme-linked immunosorbent assay (ELISA), FCS and quantitative real-time reverse transcription/polymerase chain reaction (RT-PCR) were then employed to monitor the passage-dependent effects on transgene expression and to estimate the total β-actin/pEYFP-actin ratio. NK1-R was characterized via radioactive ligand binding and the second messenger assay. The suitability of the pEYFP-actin as a marker of endogenous actin was assessed by colocalizing pEYFP-actin with rhodamine-phalloidine-stained F-actin and by comparing receptor- and jasplakinolide-induced changes in the actin cytoskeleton organization. These experiments demonstrated that: i) both constructs expressed in the generated transfected cell line are functional; ii) the estimated pEYFP-actin: endogenous β-actin ratio is within the limits required for the functional integrity of the actin filaments; and iii) pEYFP-actin and rhodamine-phalloidine-stained F-actin structures colocalize and display comparable reorganization patterns in pharmacologically challenged cells.
seven transmembrane receptor
- Bmax :
max. receptor number
bovine serum albumin
Dulbecco’s modified Eagles’s Medium
Dulbecco’s phosphate-buffered saline
enzyme-linked immunosorbent assay
flow cytometry analysis
flow cytometry sorting
green fluorescent protein
G-protein coupled receptor
N-terminally HA-tagged human neurokinin-1 receptor
human embryonic kidney cells
heat inactivated fetal calf serum
- IC50 :
50% inhibitory concentration
inositol phosphate 1
messenger ribonucleic acid
neurokinin type 1 receptor
enhanced yellow fluorescent protein
quantitative real-time reverse transcription / polymerase chain reaction
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Hrovat, A., Zavec, A.B., Pogačnik, A. et al. Establishing and functional characterization of an HEK-293 cell line expressing autofluorescently tagged β-actin (pEYFP-ACTIN) and the neurokinin type 1 receptor (NK1-R). Cell Mol Biol Lett 15, 55 (2010). https://doi.org/10.2478/s11658-009-0034-0
- Actin filaments
- Neurokinin type 1 receptor
- Flow cytometry
- Confocal microscopy