Figure 5. The SD of IP₃R1 causes aggregation of beads.

(A) Silver-stained gel using equivalent amounts of material shows the proteins present in the incubation used to prepare SVA beads (input), the unbound protein (supernatant), the washings from the beads (wash), and the proteins eluted from the beads (eluate, 85°C for 10 min). (B) Summary results show the percentages of protein immobilized on the beads for NT, IBC and SD. (C) Equilibrium competition binding to NT-biotin and IBC-biotin with ³H-IP₃ (0.75 nM). (D) Functional protein quantified from the amount of ³H-IP₃ (mol) bound per mol of protein. Results (C and D) are means ± SEM, n = 3. (E) DIC images of the indicated beads. Scale bar = 20 µm. (F) Summary results (means ± SEM, from 3 independent experiments each with 5 fields). *P = 0.041 relative to NT-beads. (G) Western blot, probed using HRP-conjugated streptavidin, shows purified samples of SD-biotin (lane 1; ∼27 kDa), IBC-biotin (lane 2; ∼44 kDa) and NT-biotin (lane 3; ∼70 kDa). Lane 1 is from a separate blot. The protein preparations did not contain detectable amounts of residual GST-tagged SD, IBC or NT fragments which have predicted sizes of 54 kDa, 71 kDa and 96 kDa, respectively. (H) The SD (left) or NT (right) interact and thereby cause beads to aggregate, whereas beads coated with IBC (centre) do not aggregate.