Characterization of AdPDE3B-overexpressed PDE3B in INS-1 (832/13) cells.
A. INS-1 (832/13) cells were infected with either Ad-βgal or AdPDE3B virus for 2 hours, 16 hours prior to experiment. Cells were homogenized and analyzed by PDE3 activity assay or immunoblot analysis using anti-PDE3B antibody. B. Immunoisolation of recombinant 32P-labelled PDE3B from AdPDE3B infected INS-1 (832/13) cells. A crude membrane fraction was used as starting material for immunoprecipitation using anti-PDE3B antibody or anti-flag M2 gel. Thoroughly washed immunoprecipitates were run on SDS-PAGE and transferred to nitrocellulose membranes. Membranes were subjected to autoradiography, immunoblot analysis using anti-PDE3B antibodies and Ponceau S staining, respectively. C. Adβ-gal or AdPDE3B-infected INS-1 (832/13) cells were subjected to subcellular fractionation using density gradient centrifugation (Percoll™). Cytosol (Cyt), granule (Gran) and plasma membrane (PM) fractionations were prepared and analyzed with immunoblot analysis for PDE3B expression. The purity of the fractions was evaluated by immunoblot analysis of the plasma membrane marker Na+/K+-ATPase (n = 2). D. Cells were AdPDE3B-infected, pre-incubated in low glucose for 2 hours and then stimulated with 16 mM glucose, 100 nM insulin or 60 mM K+ for 1 hour. Cells were homogenized and PDE3 activity was measured (n = 3).