Measurements of organelle free Ca2+ in PMR1-depleted cells. PMR1 siRNA-transfected (♦) or control scrambled siRNA-transfected (□) INS1 cells were infected with
Cyt.Aq-encoding (A–C), VAMP.Aq-encoding (D and G), or ER.Aq-encoding (E, F, and H) adenoviruses 24 h after siRNA transfection. A, B, and D–H: After a further 24 h, cells were depleted of Ca2+, the aequorin reconstituted in Ca2+-free KRBB, and then cells were perifused with KRBB supplemented with 1 mmol/l EGTA. In intact cells, 1 mmol/l EGTA was replaced
with 1.5 mmol/l CaCl2 where indicated. G and H: Cells were permeabilized with 20 μmol/l digitonin in intracellular buffer containing 1 mmol/l EGTA (free [Ca2+] <1 nmol/l) and then, where indicated, free [Ca2+] was increased to 400 nmol/l using an EGTA-buffered Ca2+ solution. C: At 24 h after adenoviral infection, cells were reconstituted with coelenterazine in KRBB containing 1.5 mmol/l CaCl2. Cells were perifused in the same medium before stimulation with 50 mmol/l KCl. In all experiments, cells were finally lysed
in hypotonic medium containing 100 μmol/l digitonin and 10 mmol/l CaCl2. In all cases, data are the means of four separate experiments.