Journal of Signal Transduction / 2015 / Article / Fig 3

Research Article

Analysis of AKAP7 Dimerization

Figure 3

Mapping the sites in AKAP7γ responsible for oligomerization. (a) Schematic depicting AKAP7γ fragments used for pulldown experiments. (b) Lysates from AKAP7γ-EGFP transfected HEK-293 cells were subjected to pulldown assays using bacterially purified S-tagged AKAP7 deletion fragments precharged on S-protein resin; AKAP7γ-150–323, AKAP7γ-1–150, full length AKAP7γ, or AKAP7α. Anti-GFP antibody was used for detecting protein interactions by western blot analysis (upper panel). Protein stain of the nitrocellulose membrane before western blot analysis is shown in the lower panel. Input from the transfected cells (20 μL) is shown in the first lane; . (c) The full length AKAP7γ sequence was spotted as overlapping 20-mer peptides with 3 amino acid shifts on cellulose membranes and subjected to overlay using S-tagged AKAP7γ. Binding was detected by chemiluminescence using an HRP-conjugated S-protein antibody (upper panel). Underlined sequences indicate regions of highest affinity binding. The antibody did not detect any binding in the absence of S-tagged AKAP7γ overlay (lower panel); . (d) In vitro pulldown of purified S-tagged AKAP7γ (10 μg) incubated with AKAP7γ-MBP precharged on amylose agarose resin (lanes 2 through 9) with increasing concentrations of either AKAP7γ-1–100 (lanes 4-5), AKAP7γ-150–300 (lanes 6-7), or both (lanes 8-9). Anti-S-tag antibody was used to detect the interaction of AKAP7γ with AKAP7γ-MBP (upper panel). The competing fragments are shown in the lower panel. Middle panel shows the protein stain of the nitrocellulose before western blot analysis to demonstrate equal amounts of MBP-tagged proteins. (e) Normalization of binding shown in (d).
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