Fig. 1. The substrate-trapping PTP1B DA localizes to punctate structures at cell margins. Cells were plated on fibronectin-coated coverslips, fixed and examined by wide-field fluorescence microscopy. (A) PTP1B null cells were transfected with GFP-wild-type PTP1B or (B) GFP-PTP1B DA and visualized 20 hours later. The arrows indicate punctate structures (B, inset). Note the absence of punctate structures in A. (C) PTP1B null cells stably reconstituted with PTP1B DA were stained with monoclonal anti-PTP1B followed by Alexa Fluor 488-conjugated secondary antibody. Note that the punctate structures look larger because of the signal amplification by the antibodies. Bar, 20 µm. (D) Representative western blot of equivalent amounts of soluble protein prepared from PTP1B null cells (lane 1); PTP1B null cells transiently transfected with GFP-wild type PTP1B (lane 2); GFP-PTP1B DA (lane 3); or stably reconstituted with PTP1B DA (lane 4). The endogenous levels of PTP1B in a non related fibroblast cell line is also shown (lane 5). The blot was probed with anti-PTP1B.