Figure 1

Docking of AKAP5 to full-length AKAP12 as well as to N-(1-840) and C-(840-1782) terminal fragments. A, Purified HA-tagged AKAP12 was immobilized to agarose beads, His-tagged AKAP12 (1-840) or His-tagged AKAP12 (840-1782) were cross-linked individually to CNBr-activated Sepharose 4B beads. Each of these bead types was incubated with either purified His-tagged AKAP5 or an equivalent amount of purified GST-tagged G3BP1 protein, as a control. At the end of the incubation the beads were collected from the mixtures and washed. The AKAP5 released from binding to sequences immobilized to the beads (Pull down), the AKAP5 input to the incubation (Input), and the AKAP5-depleted supernatants of the incubation post affinity adsorption (Supernatant) were sampled and applied to SDS-PAGE, subjected to immunoblotting and stained with anti-AKAP5 (upper panel) or anti-GST (lower panel) antibodies. B, A431 cells stably expressing GFP-tagged AKAP12 were transiently transfected with HA-tagged AKAP5. Confluent cells were treated with 10 μM isoproterenol for 10, 20, 30 min or treated for 30 min with isoproterenol and then washed-free of agonist for 2 h (wash-out 2 h). Whole-cell lysates were prepared and pull-downs conducted with anti-HA antibody (targeting HA-tagged AKAP5) or control IgG. The immune complexes were subjected to SDS-PAGE, transferred to PVDF membrane and probed with antibodies against HA tag (HA-AKAP5) or GFP (AKAP12-GFP). The results shown are taken from a single experiment, representative of at least three separate experiments performed on separate cultures of cells.