Properties of the NT and IBC used for FP assays. The constructs used showing
the N-terminal GST tag used for purification, the PreScission cleavage site
(arrow), and the N terminus of the protein after cleavage with the residual
non-native residues underlined (A). Silver-stained gel (left) and Western
blot using antipeptide antiserum to residues 62 to 75 (right) for purified
NT (4 μg, 5.1 pmol) (B). Silver-stained gel (left) and
immunoblot with antipeptide antiserum to residues 326 to 343 (right) for
purified IBC (4 μg, 1.7 pmol) (C). Gels and blots (B and C) are
typical of at least three analyses. Positions of molecular mass markers
(kDa) are shown alongside each gel. Saturation binding of
[3H]IP3 to the NT determined in TEM (30 ng total
protein) shown as a Scatchard plot (D). Equilibrium competition binding of
IP3 and [3H]IP3 (0.75 nM) to the NT in
TEM (150 ng total protein) and CLM (4 μg total protein) (E).
Results (D and E) show means ± S.E.M., n =
3.