Fig. 3

Ca²⁺ responses in tpc2⁻cells. a Ax2- and tpc2-null cells were developed at a density of 4 × 10⁵ cells/cm² in HKC-LoCa buffer for 6 h (T6). The cells were stimulated with 50 nM cAMP at time 0. The [Ca²⁺]c level was recorded as YFP/CFP ratio images using a widefield fluorescence inverted microscope. The time (seconds) relative to cAMP addition is shown. Scale bar, 50 μm. One experiment representative of three. b In three individual experiments as described in A, the mean ratio was calculated at the times shown and the change in ratio compared to the cells 40 s prior to cAMP addition plotted (mean + SEM). c Vesicles were isolated from Ax2 and tpc2⁻ cells developed in shaking suspension in KK2 and EGTA for 4 h, with pulses of cAMP (50 nM) added every 6 min; 200 μg of the vesicles was added to 100 μl Ca²⁺ uptake buffer supplemented with 100 μM NaN₃, 1.5 mM ATP and 6 μg/ml oligomycin A. Representative trace showing calcium uptake by vesicles made from Ax2 and tpc2⁻ cells and the average gradient of uptake from the three independent experiments. d mRNA was extracted from Ax2 or tpc2⁻ cells growing or developed in shaking suspension for 2 h and levels of patA expression quantified by qRT-PCR relative to expression of IG7. Results are the average of two independent experiments (mean + SEM)