Figure 4.

Optimization of assay conditions II: divalent cation, ATP, or GTP dependence. The donor membrane was prepared from 3T3-L1 adipocytes expressing mycGlut4 treated with 10⁻⁷ M insulin for 2.5 min (IDM). The PM was prepared from nontransfected 3T3-L1 adipocytes treated with 10⁻⁷ M insulin for 5 min (IPM). The assays were performed as described in Fig. 3. (A) The assay was performed by using IPM and IDM in the presence of the indicated concentration of Ca²⁺. Note that, in all cases, the assay mixture also included 0.5 mM EGTA. (B) The assay was performed in the presence and absence of 1 mM ATP, 1 mM GTP, or 100 nM wortmannin (Wort) as indicated.