Figure 1

Expression and functional equivalence of AKAR2-AKAP5 fusion protein to native AKAP5. HEK293 cells were transfected with an expression vector harboring one of the following: either AKAR2; AKAP5; AKAR2-AKAP5 or AKAR2(T/P)-AKAP5. Cell lysates were analyzed by SDS-PAGE and resolved proteins transferred to nitrocellulose membranes. Blots were probed with antibodies specific for AKAP5 or GFP (A). HEK 293 cells were transfected with antisense morpholino oligonucleotides (AKAP5 morph) targeting the 5′ untranslated region (UTR) of human AKAP5. At 24 h later, AKAP5-deficient cells were divided into two batches: one was transfected with empty plasmid; and the other transfected with plasmid encoding AKAR2-AKAP5 fusion protein. AKAP5-deficient cells and wild-type cells each were stimulated with 10 μM isoproterenol (ISO). Activation of the mitogen-activated kinases ERK1,2 was established by activation- and phospho-specific antibody staining of immunoblots. Blots were probed with antibodies specific for either activated- and phosphorylated- pERK or pan-ERK (C). The efficiency of the antisense morpholinos used to suppress endogenous AKAP5 in these experiments is shown (B). β-actin is used as internal control for loading equivalence and was stained with anti-actin antibodies. The changes in ISO-stimulated activation of ERK in celles in which AKAP5 was knocked-down (KD) and then rescued by exogenous expression of AKAR2-AKAP5 as displayed in Panel C are plotted (D). The results displayed are mean values and s.e.m. derived from at least three separate experiments. *, denotes significance of p < 0.05.