Fig. 2.
Effect of increased SET protein on calcium mobilization by the Gq-coupled M3 muscarinic and P2 purinergic receptors in CHO-M3 cells. Top panel, 12 μg of protein homogenates from CHO-M3 cells transfected with pcDNA3 or pcDNA3::His-SET were electrophoresed on denaturing polyacrylamide gels (10%) and probed with anti-SET antibody. Bottom panel, transfected CHO-M3 cells were plated in 96-well black plates precoated with poly-d-lysine and loaded with fluorescent dye. Cells were stimulated with increasing concentrations (10−9–10−5 M) of the muscarinic agonist, carbachol (left panel) or the P2 purinergic agonist, UTP (right panel) and intracellular calcium mobilization was measured. Excitation fluorescence was 485 nm, and emission was detected at 520 nm using a 515 nm emission cutoff filter, and fluorescence emissions were measured with the FlexStation III (Molecular Devices). The increases in intracellular calcium were determined by subtracting the baseline to peak values (heights). Results were expressed as the percentage of the maximal response in cells transfected with pcDNA3 (control). The data are presented as the mean ± S.E.M. of two to four independent experiments each performed in triplicate.