Technical Reference #1930

1930.

C-terminal processing of GABARAP is not required for trafficking of the angiotensin II type 1A receptor Jawed Alam; Dawn DeHaro; Kevin M. Redding; Richard N. Re; Julia L. Cook, Ochsner Clinic Foundation, Regulatory Peptides, 159(1930), (2010)
Link To Paper

Keywords:
Protein binding; Yeast two-hybrid; GPCR; cAMP signaling pathway; Autophagy

Materials & Methods:
Rat adrenal pheochromocytoma PC-12 (ATCC CRL 1721) cells were cultured in a humidified atmosphere (95% air 5% CO2) at 37 °C in RPMI 1640 medium supplemented with 10% heat-inactivated horse serum 5% fetal bovine serum (FBS) and 50 ng/ml gentamicin. Transient transfection was carried out using Lipofectamine 2000 transfection reagent (Invitrogen) according to the manufacturer's recommendation. For immunoblot analysis cells were seeded (1×106/60 mm plate) and transfected 24 h later with 5 μg of the expression plasmid DNA. The transfection media was removed 6 h later and the cells were cultured for an additional 42 h in standard growth media. Harvesting of cells extract preparation and immunoblot analysis were carried out as previously described [4]. For imaging cells were seeded (1×106/plate) on 35 mm MatTek (Ashland MA) glass bottom culture dishes and transfected 24 h later (~70–80% confluence) with a DNA mixture consisting of 1 μg each of plasmids encoding fluorescent or control proteins. 3D deconvolution microscopy was carried out 24–48 h after transfection as previously described [4]. For reporter gene analysis cells were seeded (1×105/ well of 12-well plate) and transfected 24 h later with a DNA mixture consisting of (per well): 133 ng of pCRE-luc 100 ng of pCMV/β-gal 133 ng pCMV/myc/AT1R and 400 ng of pCMV/HA/GABARAP or the corresponding mutants or the empty vector. After 24 h the media was replaced with fresh media containing 0.5% FBS and the cells were cultured for an additional 24 h. Cells were treated for 5 h with vehicle or 100 nm Ang II in fresh culture media supplemented with 0.5% FBS and then harvested for enzyme assays. Preparation of cell extract and measurement of luciferase and β-galactosidase activities were carried out as previously described [15].

Microscopic Technique
3D deconvolution microscopy

Cell Type(s)
Rat adrenal pheochromocytoma PC-12 cells