Figure 6

Oligomerization of AKAP5 in vivo and in vitro. (A) HEK293 cells stably expressing GFP-tagged AKAP5 were transiently transfected with HA-tagged AKAP5. Cell lysates were subjected to pull-downs mediated by IgG (control) or anti-HA antibodies (IP-HA) and the pulldowns subjected to SDS-PAGE, immunoblotting, and stained with anti-AKAP5 antibodies. Endogenous AKAP5 (expressed at <10% of that of exogenously expressed, tagged-AKAP) display a M _r identical with the HA-tagged version under these conditions. (B) c-Myc-tagged AKAP5 fused to amino acids 1-147 of the GAL4 DNA-binding domain (BD) was expressed in yeast. The c-Myc tagged AKAP5 was subjected to pull-down with anti-c-Myc antibody or control IgG. The immune complexes then were incubated with purified His-tagged AKAP5. The association of His-tagged AKAP5 with the c-Myc-tagged AKAP5 was detected by SDS-PAGE, immunoblotting, and staining of the blots with anti-AKAP5 antibodies. (C) HEK293 cells stably expressing GFP-tagged AKAP5 were transiently co-transfected with an expression vector harboring HA-tagged AKAP5. The cells were either untreated or incubated with a chemical inhibitor for either MEK1/2 (PD98059, 20 μM) or cyclic AMP phosphodiesterase 4 (Rolipram, 10 μM) for 45 min prior to cell lysis and analysis by SDS-PAGE. Pull-down of the HA-tagged AKAP5 was accomplished with anti-HA antibodies. The immune precipitates were subjected to SDS-PAGE and immunoblotting. The resolved, transferred protein blots were stained with anti-AKAP5 antibodies. The results displayed are representative of at least three separate experiments performed on as many separate cell cultures.