Loss of protein tyrosine phosphatase activity and protein from A431 cells by UV irradiation. (A) Hypersensitivity of PTP1B to UV irradiation. Confluent A431 cells were irradiated with different doses of UVA. PTP1B was immunoprecipitated from lysates, and its activity was measured using the pNPP assay. Alkaline phosphatase (AP) and alcohol dehydrogenase (ADH) activities were assayed as described in Methods, and the activity of caspase 3 was assayed using a commercial kit (Calbiochem). (B) Reversible inactivation of PTP1B. PTP1B immunoprecipitates obtained as in (A) were split, either mock-treated or treated with 50 mM NAC at 25°C for 10 min under agitation, and activity was determined by the pNPP assay. (C) The data shown in (B) were normalized for the protein amounts of PTP1B in the immunoprecipitates as detected by immunoblotting. (D) Loss of PTP protein on UV irradiation. Within 1–2 min after irradiation, cells were lysed and subjected to western blotting using antibodies directed against PTP1B, SHP1, LAR-PTP and actin. (E) No loss of PP-2A, pro-caspase 8, actin or Hsp90 occurred on UV irradiation. Immunoblots are as in (D).