TABLE 4.

Inhibition of pp125^FAK by neutralizing antibody in the immune complex kinase assay and adipocytes^a

Parameter	Phosphorylation (% of basal)
	Antirat IgG			Anti-pp125FAK
	Basal	PIG 41	Insulin	Basal	PIG 41	Insulin
Phosphorylation
Cell-free paxillin	100			33 ± 8
Cellular pp125FAK	100	1,345 ± 210	576 ± 102	86 ± 10	711 ± 95	342 ± 48
Cellular paxillin	100	635 ± 102	289 ± 45	93 ± 7	295 ± 42	105 ± 18
Cellular IRS-1	100	1,487 ± 255	1,753 ± 188	89 ± 5	804 ± 92	1,693 ± 140
Glucose transport	100	1,777 ± 142	1,896 ± 153	90 ± 8	925 ± 48	1,958 ± 166

^aDefatted cell lysates from isolated rat adipocytes were incubated (30 min, 4°C) in portions (10 μg of protein) with neutralizing anti-pp125^FAK or antirat IgG (IgG) antibody (1:100). After immunoprecipitation with monoclonal anti-pp125^FAK antibody, the immune complexes were subjected to kinase assay by incubation with [³²P]ATP in the presence of paxillin. Total mixtures were separated by SDS-PAGE. Phosphorylated paxillin was quantitatively evaluated by phosphorimaging. Isolated rat adipocytes were electroporated with neutralizing anti-pp125^FAK or antirat IgG antibody (1:50) and then incubated (15 min, 37°C) in the absence or presence of PIG 41 (3 μM) or human insulin (10 nM). pp125^FAK, paxillin, and IRS-1 were immunoprecipitated with the corresponding antibodies from defatted cell lysates and then immunoblotted with antiphosphotyrosine antibody. Phosphorylated proteins were quantitatively evaluated by phosphorimaging. Portions of the cells were assayed for 2-deoxyglucose transport. Basal values (incubation and electroporation with IgG) were set at 100% in each case. The experiment was repeated three times (mean ± standard deviation).