Table 1.

Inhibition of pp59Lyn by neutralizing antibody in the immune complex kinase assaya

Antibody dilutionPhosphorylation (arbitrary units)
AutoEnolaseIRS-1
None15,899 ± 1,7562,974 ± 4356,452 ± 855
Anti-rat IgG (1:50)14,247 ± 1,5303,012 ± 2777,231 ± 1,034
Anti-pp59Lyn
 1:10,00015,023 ± 1,9803,189 ± 3556,974 ± 893
 1:2,00013,577 ± 1,4362,746 ± 3014,760 ± 599
 1:5005,231 ± 9651,235 ± 1881,921 ± 266
 1:1001,930 ± 214702 ± 871,213 ± 158
 1:50895 ± 101254 ± 331,045 ± 213
 1:25799 ± 132212 ± 45823 ± 174
  • a pp59Lyn was immunoprecipitated with monoclonal anti-pp59Lyn antibody from defatted cell lysates (portions of 5 μg of protein) prepared from isolated rat adipocytes. The immune complexes were then incubated (30 min, 4°C) with neutralizing anti-pp59Lyn or anti-rat IgG antibody at various dilutions and subsequently subjected to kinase assay by incubation with [32P]ATP in the absence (for autophosphorylation) or presence of denatured enolase or IRS-1. Total mixtures were separated by SDS-PAGE. Phosphorylated pp59Lyn, enolase, or IRS-1 was quantitatively evaluated by phosphorimaging (arbitrary units). The experiment was repeated three times (mean ± standard deviation).