Fig. 3. PTP1B puncta co-localize with Src and Src-pY418 at cell margins. PTP1B null cells were transfected with GFP-PTP1B DA and examined 20 hours later by fluorescence microscopy (A-G). Cells were labeled with anti-Src (A-F) or anti-Src-pY418 (G) followed by a secondary antibody conjugated with Alexa Fluor 568. Note the co-localization (yellow) at large punctate structures formed at the peripheral ER network (C), or in small punctate structures at individual ER tubules at the cell margins (F) Arrows indicate puncta. Note that a subpopulation of PTP1B DA puncta is coincident with the phospho-active form of Src (G). Bars, (A-C) 10 µm; (D-G) 15 µm. (H) The formation of Src-PTP1B complexes was analyzed by immunoprecipitation. PTP1B null cells (-/-), and cells stably expressing PTP1B wild type (Wt) or PTP1B DA (DA) were lysed and immunoprecipitated with anti-PTP1B. The amount of PTP1B and Src in the lysate (input) and in the immunoprecipitates was detected with the respective antibodies (Blot).