Journal of Signal Transduction / 2015 / Article / Fig 4

Research Article

Analysis of AKAP7 Dimerization

Figure 4

PKA and PLB binding to AKAP7γ do not interfere with AKAP7γ dimerization. (a) Lysates from either AKAP7γ-EGFP or AKAP7γ-ΔPKA-EGFP transfected HEK-293 cells were subjected to pulldown assays using bacterially purified S-tagged AKAP7γ proteins precharged on S-protein resin; full length AKAP7γ or AKAP7γ-1–268 (which lacks the PKA binding domain). 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 middle panel; . (b) Lysates from AKAP7γ-mCherry transfected HEK-293 cells were subjected to pulldown assays using bacterially purified S-tagged AKAP7γ precharged on S-protein resin. After an overnight incubation, the pulldowns were washed extensively and then incubated with the PKA anchoring disrupting peptide AKAPIS or control, scrambled peptide (10 μM) for 3 hours. The pulldowns were washed again, and association of AKAP7γ was determined by anti-mCherry antibody (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 middle panel; . (c) The AKAP18γ peptide array membranes used in (c) were stripped for 45 min in 62.5 mM Tris-HCl, pH 6.8, 2% SDS, and 100 mM β-mercaptoethanol at 60°C and washed three times in TBST for 10 min before being blocked in 1% casein (in TBST) overnight at 4°C. Concomitantly, 2 µg/mL of recombinant human His-S-AKAP18γ protein was preincubated with or without 10 µM phospholamban peptide (1–30) overnight at 4°C, before being overlaid onto the peptide array membranes for 2 hours at room temperature. The membranes were washed, incubated with HRP-conjugated anti-S-tag antibody, and developed as described above.
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