Fig. 3. Up-regulation of PMCA mRNA, protein and activity in SERCA2^+/– cells. In (A–C), intact pancreatic acini from wild-type (A and C) or SERCA2^+/– mice (B and C) were added to a Ca²⁺-free, high K⁺ solution containing ∼7.5 µM EGTA and 2 µM of the free acid form of Fura 2 (see Materials and methods). Where indicated by the arrows, the cells were stimulated with 1 mM carbachol. The rate of unidirectional Ca²⁺ efflux was calculated from the first derivative of the slopes and corrected for the basal Ca²⁺ efflux prior to agonist stimulation. (D) RT–PCR analysis of PMCA isoforms in mRNA prepared from brain or pancreatic acini. In the case of the pancreas, the products of PMCA1 and 4 obtained in the first round of amplification were used as the template for a second round of amplification, and the products of the second round are shown in the last two lanes. For the western blots in (E) and the RT–PCR analysis in (G), brains were collected from six wild-type and six SERCA2^+/– mice. Parts of the brains were homogenized immediately to prepare brain microsomes and then brain extracts. A second portion of the brains was used to extract the mRNA. The blot in (E) was probed with the 5F10 antibody and the results were analyzed by densitometry (F). (H and I) Confocal images of immunolocal ization of PMCA using the 5F10 antibody that recognizes all PMCA isoforms (H) and the JA9 antibody that specifically recognizes PMCA4 (I).