In vitro binding to the SH3 domain of ht-HckSH3 by SF2Nef and SF2Nef mutants. Purified GST and GST-SF2Nef fusion proteins were incubated with the Hck SH3 domain at 3 μM each. The extent of Hck binding to GST or GST-SF2 fusion proteins was assayed as previously described . Upper Panel, Bound His-tagged-Hck SH3 domain protein (ht-HckSH3) was determined by Western blot analysis with anti-His tag antibody. After GST-F90A, a lane was skipped and 20% of the amount of ht-HckSH3 was added to the binding reactions and was run in the last lane. Lower Panel, Equal amounts of GST and GST fusion proteins were added to the binding assays as shown by SDS/PAGE followed by Coomassie Blue staining.