Figure 2.

GAIP_80–206 is responsible for its GAP activity. Recombinant GAIP fragments (2.0 μM) were added to [γ-³²P]GTP-loaded recombinant Gα_i3 obtained by size-exclusion chromatography (G50) in the presence of 15 mM MgSO₄. Aliquots were taken at the indicated times, and reactions were stopped with a charcoal slurry. Both full-length GAIP_1–217 (▴) and the RGS domain, GAIP_80–206 (●), stimulate the GTPase activity of Gα_i3. No activity was seen after boiling. (▵), GAIP_1–217; (○) GAIP_80–206. (Inset) GAIP (C) antibody inhibits GAP activity of full-length GAIP. GAIP_1–217 was preincubated with 10 μl of GAIP (C) (■) or CALNUC (□) antiserum, diluted as indicated, and added to Gα_i3. Reactions were stopped after 2 min of hydrolysis. GAP activity was dose-dependent and inhibited by GAIP (C) antiserum (3–100 nM). Data shown are representative of three experiments.