Fig. 1.
SET siRNA knockdown in CHO-M3 cells and effect of SET knockdown on calcium mobilization by the Gq-coupled M3 muscarinic and P2 purinergic receptors. A, 12 μg of protein homogenates from CHO-M3 cells transfected with SET siRNA oligonucleotides 268–291 or SET siRNA oligonucleotides 327–351 and 530–554 and their controls (C and C*, control siRNA with 12 or 2 mismatches from the siRNA SET, respectively) were electrophoresed on denaturing polyacrylamide gels (10%) and probed with anti-SET or anti-actin antibodies. B, CHO-M3 cells transfected with SET siRNA (327/530 or 268) or the respective controls were plated in 96-well black plates precoated with poly-d-lysine and loaded with fluorescent dye. Cells were stimulated with increasing concentrations (10−9–10−4 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; fluorescence emissions were measured with the FlexStation 3 (Molecular Devices). The increases in intracellular calcium were determined by subtracting the baseline to peak values (heights). Results are expressed as the percentage of the maximal response in control cells (control). The data are presented as the mean ± S.E.M. of three independent experiments in triplicate.