Cos7 cells were used when pSVL expression plasmids were used because proteins encoded by pSVL plasmids express better in Cos7 cells than in HEK293T cells. HEK293T cells were used for all other experiments. Ten-centimeter plates of cells were washed twice with cold phosphate-buffered saline (PBS) and scraped into 600 μl of phosphatase inhibitor buffer [25 mM Hepes (pH 7.4), 20% glycerol, 50 mM NaCl, 50 mM NaF, 2 mM EDTA, 0.5 μM microcystin, and 1× Roche cOmplete EDTA-free Protease Inhibitors]. Cells were lysed by sonication at 40% power (Misonix Sonicator XL) for 5 s, and then the insoluble membrane fraction was pelleted by centrifugation at 25,000g for 15 min. The supernatant was aspirated and membranes washed with 400 μl of phosphatase inhibitor buffer before being resuspended in phosphatase inhibitor buffer for the GC assay. Activity assays were performed as described previously (38) using 20 μl of prepared crude membranes, 20 μl of a 5× activation mix containing the divalent metal (5 mM MgCl2 or MnCl2 at reaction volume), and any other activators used [1 mM ATP, 1 μM ANP, 1 μM CNP, or 1% (v/v) Triton X-100 at reaction volume] and adding 60 μl of prewarmed reaction cocktail to final reaction conditions of 25 mM Hepes (pH 7.4), 50 mM NaCl, 500 mM isobutyl-methyl-xanthine, 0.5 μM microcystin, 1 mM EDTA, 0.1% bovine serum albumin, and 5 mM creatine phosphate and creatine kinase (0.1 μg/ml) as a nucleotide triphosphate regeneration system. If not otherwise indicated, GTP concentrations were 0.1 mM. Substrate-velocity assays used GTP concentrations ranging from 0 to 3 mM. Time course assays used time points at 10 s, 2 min, and 10 min. Cyclic GMP content in single-substrate concentration assays was determined by radioimmunoassay (56) or by ELISA by diluting NaOAc-buffered GC assay samples in 0.1 N HCl and following the manufacturer’s instructions. cGMP content in substrate-velocity assays was determined by radioimmunoassay as previously described (24) or by purification followed by ELISA estimation of cGMP concentrations. Substrate-velocity assays tested by ELISA were terminated in 110 mM ZnOAc and 110 mM Na2CO3 and purified on acidified alumina as previously described (68) with the exception of eluting in 3 ml of 200 mM ammonium formate to separate ATP and GTP from cGMP. Eluent was used undiluted in ELISA measurements according to the manufacturer’s instructions with the exception of being performed in 200 mM ammonium formate rather than 0.1 N HCl. Data were normalized to a control value [Vmax for GC-A-WT (+ATP)] in Fig. 8 due to variations in transfection efficiency. To remove this confounding error associated with transfection efficiency and preserve all differences due to the treatments or mutations, we expressed all activity data in Fig. 8B as a percentage of the Vmax for GC-A-WT (+ATP).

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