Figure 3.

GAIP is associated with intracellular membranes in pituitary cells. GAIP-containing fractions 3–7 prepared from AtT-20 cells (see Figure 2) were pooled and diluted in PBS with protease inhibitors (chymostatin, leupeptin, antipain, and pepstatin A), and 1.0-ml aliquots were incubated at 4 C overnight with WGA-agarose (400 μg/ml) to deplete them of PM. Bound (B) and nonbound (NB) membranes were assayed for 5′-nucleotidase activity (upper panel) and solubilized and immunoblotted for GAIP (lower panel). Most (80%) of the 5′-nucleotidase activity is found in the bound fraction. GAIP is found in the nonbound fraction. Data are means ± SD of three independent experiments.