Table 2.

Specificity of the antibodies used for neutralization and immunoprecipitation of pp59Lynand pp125FAKa

AntibodyResponse to recombinant kinase (concn of [32P]enolase or [32P]paxillin/kinase protein [% of maximum])
pp59Lynpp60Fynpp60Srcpp125FAKpp116PYK2
None100100100100100
Neutralizing
 Anti-pp59Lyn 22 ± 893 ± 9 91 ± 5
 Anti-pp125FAK 98 ± 495 ± 7103 ± 630 ± 489 ± 6
Immunoprecipitation
 Anti-pp59Lyn 85 ± 9/92 ± 50/02 ± 1/3 ± 2
 Anti-pp125FAK 0/00/3 ± 14 ± 3/078 ± 8/85 ± 66 ± 1/0
  • a Recombinant kinases (1 to 5 μg) were incubated (30 min, 4°C) in the absence or presence of neutralizing anti-pp59Lyn (1:50) or anti-pp125FAK (1:100) antibodies and then subjected to kinase assay by incubation with [32P]ATP in the presence of denatured enolase (for pp59Lyn, pp60Fyn, or pp60Src) or paxillin (for pp125FAK or pp116PYK2). Alternatively, recombinant kinases (1 to 5 μg) were immunoprecipitated with monoclonal anti-pp59Lyn (5 μg/sample) or anti-pp125FAK (2 μg/sample) antibodies. The immune complexes were subjected to kinase assay (see above) or immunoblotted with corresponding antibodies to the different kinases. [32P]enolase or [32P]paxillin and immunoblotted kinase protein were quantitatively evaluated by phosphorimaging and set at 100% in a control incubation lacking antibody in each case. The experiment was repeated three times (mean ± standard deviation).