Receptor internalization was quantitatively assessed using HaloTag technology (Promega) as described previously (4 (link)). HEK293T cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM, 5919, Nissui, Tokyo, Japan) supplemented with 10% fetal bovine serum. The cells were transfected with Halo-expressing vector and labeled with the cell-impermeable Alexa Fluor 488 ligand (Promega) in Opti-MEM for 15 min at 37°C. Each inhibitor or antagonist pretreatment was for 30 min. The cells were then treated with 1 µM PACAP, 5-HT or saline, washed with phosphate-buffered saline and fixed in 4% paraformaldehyde. Cells were imaged using an FV1000D confocal microscope (Olympus, Tokyo, Japan) in sequential mode and membrane protein internalization was quantified using ImageJ software (NIH, MD, USA). To assess the internalization ratio, we defined the shape of a whole-cell (region of interest, ROI, A) and its cytoplasmic region (ROI B) by reducing the size by 5–10 pixels and then determining the fluorescence in both ROIs. The internalization ratio (%) was defined by dividing the amount of luminescence in ROI B by that in ROI A.
Halotag technology
Manufactured by Promega
HaloTag technology is a protein tagging system that allows the covalent attachment of a synthetic ligand to a target protein. This facilitates the purification, detection, and manipulation of the tagged protein within a biological system.
Automatically generated - may contain errors
4 protocols using halotag technology
1
Quantitative Assessment of Receptor Internalization
2
Cell Labeling for Live Imaging
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We labeled cells just before imaging. Following HaloTag technology protocol (Promega), cells were incubated for 30 min in DMEM 10%FCS supplemented with 0.1 nM JF 549. Then, cells were rinsed 3 times with DMEM + 10% SVF and were re-incubated for 30 min. Finally, the cell medium was replaced by L-15 medium (Life Technologies, CA, USA) before imaging, to avoid DMEM auto-fluorescence.
3
Fluorescent Labeling of Live Cells
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We labeled cells just before imaging. Following HaloTag technology protocol (Promega), cells were incubated for 30 min in DMEM 10%FCS supplemented with JF 549 at 10 nM final. Then, cells were rinsed 3 times with DMEM + 10% SVF and were re-incubated for 30 min. Finally, the cell medium was replaced by L-15 medium (Life Technologies, CA, USA) before imaging, to avoid DMEM auto-fluorescence.
4
Quantitative Assessment of PAC1R Internalization
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Internalization of PAC1R was quantitatively assessed using HaloTag technology according to the manufacturer's protocol (Promega). HEK293T cells were transfected with PAC1R-Halo-expressing vector together with β-arrestin1 siRNA, β-arrestin2 siRNA or control siRNA; the cells were then labeled with the cell-impermeable Alexa Fluor 488 ligand (Promega) in Opti-MEM for 15 min at 37°C. Clathrin-mediated endocytosis inhibitor, 250 μg/ml ConA or 15 μM Pitstop2, were pretreated for 30 min. After 30 min, the cells were stimulated with 1 nM PACAP or saline, and were then washed with phosphate-buffered saline and fixed in 4% paraformaldehyde. The cell images were obtained using FV1000D confocal microscope (Olympus) in a sequential mode and membrane protein internalization was quantified using the ImageJ software. To assess the internalization ratio of PAC1R, we defined the shape of the whole-cell (ROI A) and the cytoplasm region (ROI B) by reducing the size by 5–10 pixels and determined the fluorescence in the both ROIs. The internalization ratio (%) was defined by dividing the amount of luminescence in ROI B by that in ROI A. All analyses were performed in a blind manner.
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