Analysis of AKAP7 Dimerization

Figure 1

Direct cellular association of AKAP7γ with itself. (a) Lysates from AKAP7γ-EGFP transfected HEK-293 cells were subjected to pulldown assays using bacterially purified AKAP7γ-MBP (maltose binding protein) precharged on amylose agarose resin (lanes 1 and 2) or MBP precharged on amylose agarose resin (lanes 3 and 4). Input from the transfected cells (20 μL) is shown in lane 5. Anti-GFP antibody was used for detecting protein interactions by western blot analysis (upper panel) while the protein stain of the nitrocellulose before western blot analysis is shown in the lower panel; . (b) mCherry western blot analysis of anti-GFP immunoprecipitates isolated from HEK-293 cells cotransfected with AKAP7γ-mCherry and either EGFP alone or AKAP7γ-EGFP (upper panel). To confirm immunoprecipitation of both EGFP and AKAP7γ-EGFP, the nitrocellulose membranes were stripped and reprobed for GFP using a monoclonal anti-GFP antibody (middle panel). Lower panel depicts mCherry western blot analysis of the input from each condition; . (c) In vitro pulldown of purified S-tagged AKAP7γ incubated with AKAP7γ-MBP precharged on amylose agarose resin (lanes 1 and 2) or control MBP (lane 3). Anti-His antibody was used to detect the interaction (upper panel). Equal amounts of S-tagged AKAP7γ were used in each condition, as shown by analysis of the input used in each experiment (middle panel). Lower panel shows the protein stain of the nitrocellulose before western blot analysis to demonstrate equal amounts of MBP-tagged proteins; .

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