TABLE 1.

Inhibition of pp59^Lyn by neutralizing antibody in the immune complex kinase assay^a

Antibody dilution	Phosphorylation (arbitrary units)
	Auto	Enolase	IRS-1
None	15,899 ± 1,756	2,974 ± 435	6,452 ± 855
Anti-rat IgG (1:50)	14,247 ± 1,530	3,012 ± 277	7,231 ± 1,034
Anti-pp59Lyn
1:10,000	15,023 ± 1,980	3,189 ± 355	6,974 ± 893
1:2,000	13,577 ± 1,436	2,746 ± 301	4,760 ± 599
1:500	5,231 ± 965	1,235 ± 188	1,921 ± 266
1:100	1,930 ± 214	702 ± 87	1,213 ± 158
1:50	895 ± 101	254 ± 33	1,045 ± 213
1:25	799 ± 132	212 ± 45	823 ± 174

^app59^Lyn was immunoprecipitated with monoclonal anti-pp59^Lyn 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-pp59^Lyn 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 pp59^Lyn, enolase, or IRS-1 was quantitatively evaluated by phosphorimaging (arbitrary units). The experiment was repeated three times (mean ± standard deviation).