Abstract

Introduction

Materials & Methods

Results

Discussion & Conclusion

References

Discussion
Board

METHODS I : Assessment of Adenylyl Cyclase Activity

Assays of adenylyl cyclase activity were performed in permeabilized cells. Adenylyl cyclase activity was assessed by the conversion of [³²P]ATP to [³²P]cAMP. [³²P]cAMP was separated from [³²P]ATP by sequential Dowex and alumina chromatography and corrected for recovery with [³H]cAMP as the internal standard.

METHODS II : FAC6 Construction and Transfection

The Flag epitope DYKDDDDK was incorporated into the 5’ amino terminal end of adenylyl cyclase isoform VI (AC6) via PCR mutagenesis to generate Flag epitope-tagged AC6 (FAC6). The PCR product was directly cloned into pCR2.1 (Invitrogen) and then excised as a HindIII-XbaI insert and subcloned into the mammalian expression vector pRc/CMV (Invitrogen) to generate pRc/CMV-FAC6. HEK 293 cells were transfected via calcium phosphate precipitation method.

METHODS III : Immunoprecipitation and Immunoblotting

FAC6 was immunoprecipitated from clarified FAC6-transfected HEK 293 cell lysates using anti-Flag M2 affinity agarose beads (Research Diagnostics Inc). Immunoprecipitates were resolved electrophoretically via SDS-PAGE, and transferred from gels to PVDF membranes. Immunoblotting was carried out by incubating membranes with either anti-Flag M5 antibody, anti-AC COMM antibody, anti AC5/6 (Santa Cruz) or antiphosphotyrosine PY20 antibody (Transduction Laboratories). Immunoreactivity was detected via enhanced chemiluminescence detection. In non-transfected and sham-transfected HEK 293 cells, FAC6 was not detected (data not shown).

METHODS IV : Phosphorylation Experiments and Phosphoamino Acid Analysis

FAC6-transfected HEK 293 cells were metabolically labeled with 0.5 mCi/mL [³²P]orthophosphate and treated in the absence or presence of 300 µmol/L vanadate. Cells were lysed and immunoprecipitates were resolved via SDS-PAGE. Gels were mounted, dried and exposed to autoradiographic film.

To perform phosphoamino acid analysis, FAC6 was excised from gels and subjected to amino acid hydrolysis. Hydrolysates were spotted onto thin layer chromatography plates and subjected to two dimensional electrophoresis. Plates were then subjected to autoradiography.

METHODS V : Site Directed Mutagenesis

Candidate serine amino acid residues located in and adjacent to the C_1b catalytic region of FAC6 were mutated to alanine residues using PCR-based site directed mutagenesis.

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