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Fig. 6. In vitro phosphorylation of PTP1B by Fer. Purified, phosphatase-dead (C215S) GST-PTP1B was immobilized on glutathione-coated wells and incubated with recombinant wild-type Fer (FerWT) or kinase-dead Fer (FerD743R), and the amount of phosphotyrosine incorporated into PTP1B was measured using anti-phosphotyrosine antibodies. (A) PTP1B carrying the C215S mutation and additional mutations on tyrosine 66 (C215S/Y66F), tyrosine 152 (C215S/Y152F) or both tyrosine 66 and 152 (C215S/Y66/152F) was used as substrate and phosphorylation evaluated by ELISA using HRP-conjugated anti-phosphotyrosine antibody. (B) Immobilized kinase dead GST-PTP1B was incubated with Fer, eluted with SDS sample buffer and the amount of phosphotyrosine evaluated by western blot with anti-phosphotyrosine antibody (top). The same membrane was stripped and reprobed with anti-PTP1B antibody (bottom).
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