Figure 3.
Expression and subcellular localization of IP3R subtypes. (A) Effects of 2APB and ryanodine on the sensitization. Fura-2–loaded PC12 cells (106) were stimulated with BK (1 μM), followed by incubation with 2APB (75 μM) or ryanodine (1 μM) for 3 min, and then finally stimulated with EGF (20 nM). The peak amplitude of the EGF-induced [Ca2+]i rise was measured. Error bars are mean ± SEM of four separate experiments. **, P < 0.01. (B) In situ hybridization of the IP3R transcripts for each subtype in adrenal gland. Light micrographic images of adrenal gland sections with either antisense or sense IP3R RNA probes are shown. Bar, 400 μm. (C) Western blot analysis of adrenal medulla, PC12 cells, and CHO-K1 cells with antibodies against IP3R subtypes. (D and E) Immunoconfocal microscopy images showing colocalization of IP3R1 and flotillin-1 in adrenal chromaffin cells. Cells were triple labeled with propidium iodide (PI, red), anti–flotillin-1 antibody (blue), and with either anti-IP3R1 or anti-IP3R3 antibody (green). Immunoconfocal microscopy and postacquisition processing of images were performed as described in Materials and methods. (F) IP3R1 and PKA are present in DRMs. Extracts from adrenal medulla were fractionated by sucrose density gradient centrifugation, and each fraction was analyzed by immunoblotting with the indicated antibodies.