Figure 3

Steric-exclusion chromatography of AKAP5 and AKAP12 alone and in combination: identification of supermolecular AKAP5/AKAP12 hetero-oligomers. To ascertain the possible presence of higher-order oligomers of AKAP12 and AKAP5, steric-exclusion chromatography of purified AKAP5 and AKAP12 individually (A and B, respectively) or after incubation in combination (C) was performed on an AKTA FPLC (GE Healthcare) fitted with a HiPrep Sephacryl S-400 High resolution 16/60 column (GE healthcare). Marker proteins mobilities were employed to establish the MW of AKAP5-, AKAP12-, and AKAP5/AKAP12 supermolecular complexes by elution position. The A280 absorbance was monitored in real time. Samples from each fraction were subjected to SDS-PAGE and immunoblotting and staining for the AKAP12 or AKAP5. The results displayed are representative of at least three separate experiments performed on as many individual protein preparations.