Journal: The Journal of Biological Chemistry

Article Title: AKAP79/150 Interacts with AC8 and Regulates Ca 2+ -dependent cAMP Synthesis in Pancreatic and Neuronal Systems *

doi: 10.1074/jbc.M110.120725

Figure Lengend Snippet: Binding between AKAP79/AKAP150 and the N terminus of AC8. A , GST pull-downs using whole cell lysate from cells transiently expressing tagged AKAP proteins. Lane 1 , GST alone; lanes 2–4 , the following regions of AC8 fused to GST: 1–179 (N terminus), 582–703 (C1b domain), and 1106–1248 (C terminus), respectively. Lane 5 , input control (5%) to confirm expression of each construct. B , top , AKAP79-HA and AKAP150-HA transiently expressed in HEK293 cells were pulled down using GST-fused first half (residues 1–77) or second half (residues 73–179) of the AC8 N terminus. Bottom , immunoblotting of the GST-fused proteins used in the pull-downs in the top . The upper GST bands represent the full-length form of each protein. C , plot of densitometries from 3–6 repeats of blots presented in B to quantify binding of AKAP79/150 to GST-AC8 1–77 versus GST-AC8 73–179. Data are normalized to the intensity of the uppermost GST bands. Error bars , S.E. D , schematic diagram of GST-AC8 constructs used in A–C .

Article Snippet: For knockdown of endogenously expressed AKAP150 in primary cultured hippocampal neurons, shRNA AKAP150 lentiviral particles (Santa Cruz Biotechnology, Inc., Santa Cruz, CA) were added directly to cultured cells 72 h prior to experiments.

Techniques: Binding Assay, Expressing, Control, Construct, Western Blot

Journal: The Journal of Biological Chemistry

Article Title: AKAP79/150 Interacts with AC8 and Regulates Ca 2+ -dependent cAMP Synthesis in Pancreatic and Neuronal Systems *

doi: 10.1074/jbc.M110.120725

Figure Lengend Snippet: AKAP disruption enhances CCE-induced AC8 activity. A , CCE-mediated increases in cAMP assessed using the FRET-based sensor Epac2-camps following knockdown of endogenous AKAP79 levels. Data are plotted as relative FRET ratio changes normalized to maximum signal response. Cells were pretreated with 200 n m Tg in Ca 2+ -free conditions, and CCE was induced upon the addition of 2 m m Ca 2+ to the bath solution. B , average Fura-2 traces from HEK-AC8 cells during CCE following transfection with AKAP150-HA, AKAP79-HA, or shRNA AKAP79. All data are normalized to maximal Fura-2 signal obtained upon the addition of 5 μ m ionomycin (Ca 2+ ionophore) and 5 m m Ca 2+ . C , effects of the AKAP/PKA disruptor peptide, St-Ht31 (10 μ m ), on CCE-stimulated AC8 activity assessed using Epac2-camps. Experiments were performed in the presence of 100 μ m IBMX, and CCE was induced upon the addition of 0.5 m m Ca 2+ . St-Ht31P (10 μ m ) was used as a negative control. D , analyses of the effects of AKAP79 knockdown or pharmacological AKAP disruption (St-Ht31) on AC8 activity. Plots of peak CCE-induced cAMP increase relative to control ( left chart ) and peak rate of cAMP production ( right chart ) are presented as mean ± S.E. ( error bars ). **, p < 0.001 using Students' t test.

Article Snippet: For knockdown of endogenously expressed AKAP150 in primary cultured hippocampal neurons, shRNA AKAP150 lentiviral particles (Santa Cruz Biotechnology, Inc., Santa Cruz, CA) were added directly to cultured cells 72 h prior to experiments.

Techniques: Disruption, Activity Assay, Knockdown, Transfection, shRNA, Negative Control, Control

Journal: The Journal of Biological Chemistry

Article Title: AKAP79/150 Interacts with AC8 and Regulates Ca 2+ -dependent cAMP Synthesis in Pancreatic and Neuronal Systems *

doi: 10.1074/jbc.M110.120725

Figure Lengend Snippet: Co-immunoprecipitation of endogenously expressed AC8 and AKAP150 in MIN6 cells. A , Western blot analysis to confirm the endogenous expression of AKAP150 ( left ) and AC8 ( right ) in MIN6 cells. B , immune complexes for endogenous AKAP150 co-purify with AC8 in MIN6 cell lysate. No AC8 band is seen in IgG controls. Antibodies used for immunoprecipitation ( IP ) and subsequent immunoblotting ( IB ) were specific to AKAP150 and AC8, respectively.

Article Snippet: For knockdown of endogenously expressed AKAP150 in primary cultured hippocampal neurons, shRNA AKAP150 lentiviral particles (Santa Cruz Biotechnology, Inc., Santa Cruz, CA) were added directly to cultured cells 72 h prior to experiments.

Techniques: Immunoprecipitation, Western Blot, Expressing

Journal: The Journal of Biological Chemistry

Article Title: AKAP79/150 Interacts with AC8 and Regulates Ca 2+ -dependent cAMP Synthesis in Pancreatic and Neuronal Systems *

doi: 10.1074/jbc.M110.120725

Figure Lengend Snippet: Effects of AKAP150 on Ca 2+ -stimulated AC8 activity in MIN6 cells. A , Fura-2 data showing the standard protocol for inducing CCE in MIN6 cells. Cells were pretreated with 1 μ m Tg in Ca 2+ -free conditions for 3 min prior to the addition of 2 m m external Ca 2+ . The addition of 100 μ m 2-aminoethoxydiphenyl borate ( 2-APB ; a CCE inhibitor when used at high concentrations) from 1 min onward significantly reduced Ca 2+ entry (see bar chart inset ; ***, p < 0.001). Bi , Epac2-camps data showing the effects of AKAP150 overexpression on Ca 2+ -stimulated cAMP production compared with empty vector controls. 20 n m FSK and 100 μ m IBMX were present throughout. Data are plotted as a percentage of maximal FRET signal obtained using saturating cAMP concentrations. Bii , as above, except that the effects of lentiviral shRNA directed against AKAP150 were compared with scrambled shRNA controls. C , bar charts show mean ± S.E. values ( error bars ) for peak amplitude and rate of FRET ratio changes during CCE for data in B . *, p < 0.05 compared with shRNA controls.

Article Snippet: For knockdown of endogenously expressed AKAP150 in primary cultured hippocampal neurons, shRNA AKAP150 lentiviral particles (Santa Cruz Biotechnology, Inc., Santa Cruz, CA) were added directly to cultured cells 72 h prior to experiments.

Techniques: Activity Assay, Over Expression, Plasmid Preparation, shRNA

Journal: The Journal of Biological Chemistry

Article Title: AKAP79/150 Interacts with AC8 and Regulates Ca 2+ -dependent cAMP Synthesis in Pancreatic and Neuronal Systems *

doi: 10.1074/jbc.M110.120725

Figure Lengend Snippet: A role for AKAP150 in the regulation of Ca 2+ -stimulated AC activity in hippocampal neurons. A , the basic design of the citrine-Epac2-camps-CFP (Ci/C-Epac2-camps) sensor used for hippocampal experiments and fluorescent images taken from three individual hippocampal neurons showing cytosolic expression of the cAMP sensor. Scale bar , 20 μm. B , in vitro calibrations of the Ci/C-Epac2-camps compared with the original Epac2-camps. Note the reduced pH sensitivity of the citrine-CFP version. C , comparison of Ca 2+ -stimulated AC activity in control, AKAP150-HA-expressing, and AKAP150 knockdown hippocampal neurons assessed using Ci/C-Epac2-camps. 1 μ m FSK was added in Ca 2+ -free conditions at 120 s with the readdition of 2 m m external Ca 2+ at 180 s to monitor Ca 2+ -dependent cAMP production. Maximum FRET ratio change was obtained by the subsequent addition of 10 μ m FSK, 10 μ m isoproterenol, and 100 μ m IBMX. D , overlay of control, AKAP150 overexpression, and shRNA AKAP150 data to compare Ca 2+ -stimulated AC activities. E , data analysis showing significant delay in Ca 2+ stimulation of cAMP production in neurons overexpressing AKAP150. Data represent mean ± S.E. ( error bars ) for each condition. n values are indicated on the graph . *, p < 0.01.

Article Snippet: For knockdown of endogenously expressed AKAP150 in primary cultured hippocampal neurons, shRNA AKAP150 lentiviral particles (Santa Cruz Biotechnology, Inc., Santa Cruz, CA) were added directly to cultured cells 72 h prior to experiments.

Techniques: Activity Assay, Expressing, In Vitro, Comparison, Control, Knockdown, Over Expression, shRNA