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Figure 2 | Journal of Molecular Signaling

Figure 2

From: AKAP12 and AKAP5 form higher-order hetero-oligomers

Figure 2

Docking of AKAP5 to AKAP12. A, Purified His-tagged AKAP12 (840-1782) was cross-linked to CNBr-activated Sepharose 4B, then incubated with purified His-tagged AKAP5 under the conditions labeled on each lane: lane 1, purified AKAP5, input; lane 2, pull-down at 37°C, 1 h; lane 3, pull-down at 4°C, 17 h; lane 4, pull-down at 4°C, 1 h; and lane 5, pull-down control, Sepharose 4B beads alone. B, A431 clones stably expressing GFP-tagged AKAP5 were transiently transfected to express HA-tagged AKAP12 C-(840-1782) terminal fragment. Confluent cells were treated with 10 μM isoproterenol for the indicated times. Whole-cell lysates were prepared and pull-downs performed with antibodies against the HA tag which were covalently linked to protein A/G-agarose beads or control IgG. The immune complexes were subjected to SDS-PAGE, transferred to PVDF membrane, and probed with antibodies against either AKAP5 or the HA tag. C, A431 cells stably expressing GFP-tagged AKAP5 were transiently transfected with HA-tagged AKAP12 or the AKAP12 fragments listed (i.e., 1-362; 1-652; 654-938; and 840-1782). The whole-cell lysates were prepared and pull-downs performed with antibodies against the HA tag which were covalently linked to protein A/G-agarose beads. The immune complexes were subjected to SDS-PAGE, transferred to PVDF membrane, and probed with antibodies against either AKAP5 or the HA tag. The results shown are taken from a single experiment, representative of at least three separate experiments performed on separate cultures of cells.

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