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Fig. 7. IP3R and FKBP12 do not co-immunoprecipitate in aortic smooth muscle and neither rapamycin nor FK506 alter IP3-evoked Ca2+ increases in voltage-clamped single aortic myocytes. (A) Similar amounts of FKBP12 protein are expressed in aortic and colonic smooth muscle. Colon and aorta were each hand-homogenised as described in Materials and Methods, and solubilised supernatants from each homogenate was assayed for FKBP12 protein expression. Proteins (10 µg total) from each tissue were separated using SDS-PAGE and immunoblots were probed for the presence of FKBP12 (lanes 1 and 4). Migration and signal intensity were compared with those obtained from recombinant FKBP12 (50 ng) run alongside as positive controls (lanes 2 and 3). The blot is representative of seven and four identical experiments, in colonic and aortic smooth muscles, respectively. (B,C) IP3R1 was immunoprecipitated from solubilised aortic smooth muscle. Immunoblots were probed with rabbit anti-IP3R1 and rabbit anti-FKBP12 antibodies for the presence of (B) IP3R1 and (C) FKBP12, respectively. The first lane in each panel shows the immunoprecipitated protein from aorta, lane 2 the solubilzed preparation plus protein G without antibody, lane 3 the antibody alone and lane 4 the relevant positive control (10 µg solubilised supernatant for IP3R or 50 ng recombinant FKBP12). Arrows on the right indicate the position of molecular mass markers run in parallel to indicate protein migration on the gel. The absence of a band at the 12 kDa level (C) indicates that FKBP12 is not associated with IP3R1 in these myocytes. These data are representative of three experiments. (D,E) Photolysed caged IP3 (
) increased [Ca2+]c as indicated by F/F0. Neither (D) rapamycin (10 µM, n=3) nor (E) FK506 (10 µM; n=6) significantly altered the IP3-evoked [Ca2+]c transients (P>0.05).
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