Table 4.

Inhibition of pp125FAK by neutralizing antibody in the immune complex kinase assay and adipocytesa

ParameterPhosphorylation (% of basal)
Antirat IgGAnti-pp125FAK
BasalPIG 41InsulinBasalPIG 41Insulin
Phosphorylation
 Cell-free paxillin10033 ± 8
 Cellular pp125FAK 1001,345 ± 210576 ± 10286 ± 10711 ± 95342 ± 48
 Cellular paxillin100635 ± 102289 ± 4593 ± 7295 ± 42105 ± 18
 Cellular IRS-11001,487 ± 2551,753 ± 18889 ± 5804 ± 921,693 ± 140
Glucose transport1001,777 ± 1421,896 ± 15390 ± 8925 ± 481,958 ± 166
  • a Defatted cell lysates from isolated rat adipocytes were incubated (30 min, 4°C) in portions (10 μg of protein) with neutralizing anti-pp125FAK or antirat IgG (IgG) antibody (1:100). After immunoprecipitation with monoclonal anti-pp125FAK 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-pp125FAK 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). pp125FAK, 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).