Biochemical characterization of parental AtT-20 cells and those overexpressing Calnuc-GFP. Postnuclear supernatants (PNS) prepared from parental AtT-20 cells or those stably overexpressing calnuc-GFP or calnuc(ΔSS)-CFP were analyzed by immunoblotting. (A) Endogenous calnuc (63 kD) is found in approximately equal amounts in the cytosolic (S) (100,000 × g supernatant) and membrane (P) (100,000 × g pellet) fractions. Calnuc-GFP (91 kD) is present in both fractions but is more abundant (~80%) in the cystolic fraction. Most (79%) of the mutant calnuc(ΔSS)-CFP (89 kD) is in the cytosolic (S) fraction. (B) The distribution of POMC, intermediate, glycosylated ACTH (gACTH) and mature ACTH is similar in parental cells (NT) cells and in cells overexpressing calnuc-GF (CNG). A small amount of ACTH is found in the cytosolic fraction of cells overexpressing calnuc-GFP, presumably due to leakage during preparation of the fractions. Synaptobrevin 2 (Vamp 2), an integral membrane protein, is found exclusively in membrane fractions. 90% of the Gβ subunits and 80% of the Gαi1/2 and Gαi3 are found in membrane fractions in parental (NT) cells. A faint band of slower mobility is seen in the case of Gβ subunits in the soluble fraction of CNG cells.