Figure 1.
(A) Beads coated with interacting partners (A and B) are expected to aggregate when mixed. (B) SVA-beads coated with biotin-labelled double-stranded DNA are prepared with one population displaying a sticky end (DNA-F) and the other with a complementary sticky end (DNA-F′). Interactions between the complementary strands are expected to cause aggregation of the beads. (C) SVA beads displaying either DNA-F or DNA-F′ were prepared. Each DNA can only weakly self-dimerize with a predicted KD of ∼36 mM, while a mixture of complementary DNA can form dimers with a predicted KD of ∼63 fM (calculated using OligoAnalyzer 3.1, Integrated DNA Technologies). Differential interference contrast (DIC) microscopy was used to measure aggregation after incubation (25 µL, 30 min) of each population of beads alone (10 pmol of SVA sites and 20 pmol biotin-DNA) or after mixing (50∶50 ratio). The scale bar (20 µm) applies to all three images. (D) Summary results show the aggregation ratios (see Methods) as means ± SEM, from 3 independent experiments with 5 fields analyzed in each. *P = 0.017.
Figure 2.
Quantification of bead aggregation.
(A) The protocol used for analyzing aggregation of beads. (B–D) Differential interference contrast (DIC) images of SVA beads coated with NT-biotin showing a typical field (B), the processed image after applying an intensity-threshold (C), and an enlarged image of the box (53.6×53.6 µm) shown in C (D) with single and double arrows indicating single and double beads. Scale bars represent 20 µm in all images. (E) Cartoon representation of the constructs used. Arrows indicate the PreScission protease cleavage site and b denotes the biotinylation sequence.
Figure 3.
Immobilization of functional NT on beads.
(A) SVA-beads (60 pmol) were incubated with NT-biotin (17 pmol) and then magnetically separated from the supernatant (see Methods). The silver-stained gel shows equivalent fractions of the input (lane 2), the supernatant (lane 3), NT-beads treated (85°C, 10 min) to release bound protein (lane 4), or similarly treated control beads (lane 5). Lane 1 shows the molecular mass markers (kDa). The 70-kDa- and 15-kDa-bands correspond to NT-biotin and SVA monomer, respectively. (B) Specific 3H-IP3 binding (0.75 nM) to control or NT-coated SVA-beads. Results (dpm, disintegrations per minute) are means ± SEM, n = 3. *P = 0.019. (C) Equilibrium-competition binding to NT-biotin (black) and NT-beads (red) with 3H-IP3 (0.75 nM). Results are means ± SEM from 3 experiments.
Figure 4.
The NT of IP3R1 causes aggregation of beads.
(A) DIC images of beads prepared as indicated. (B) Summary results show means ± SEM, n = 3. *P = 0.012 relative to SVA beads. (C) Western blot, probed using HRP-conjugated streptavidin, shows equivalent fractions of the input (20 pmol of native or denatured NT-biotin, lane 1), the supernatant (lane 2), wash (lane 3) and NT-beads treated (85°C, 10 min) to release bound protein (lane 4). Lane 5 shows the biotinylated protein ladder. The ∼70-kDa bands correspond to NT-biotin. The blot is representative of two similar analyses. (D) DIC images of NT-biotin-coated SVA-beads with or without 10 µM IP3. (E) Summary results show means ± SEM, n = 5. P = 0.40. Scale bars (A and D) represent 20 µm.
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.