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. Author manuscript; available in PMC: 2018 Mar 13.
Published in final edited form as: Leukemia. 2017 Aug 18;32(3):820–827. doi: 10.1038/leu.2017.267

Figure 2. Phosphorylation of Y1196 by ABL is sufficient to elicit SOS1 RAC GEF activity.

Figure 2

A-B. MANT-GDP-fluorescence RAC GEF assay of purified pY1196F-SOS1. GST-RAC1 was equilibrated with 2 μM MANT-GDP. After 300s, we added: (B) purified, phospho-enriched SOS1 (30 µl and 120 µl, corresponding to ~ 10 and 40 nM, respectively) or 50 nM of His-DH-PH of Tiam1 (used as positive control). Buffer indicates the non-stimulated nucleotide exchange rate of RAC1: (C) purified, phospho-enriched SOS1 or un-phosphorylated SOS1 [treated with T-cell specific tyrosine phosphatase either in the presence (TCI) or the absence (TC) of T-cell phosphatase inhibitor]. The reaction containing MANT-GDP was excited at 360nm. The emission of MANT-GDP-bound to RAC was measured at 440nm. Right panels: Aliquots of phosphorylated (TCI) and phosphatases dephosphorylated (TC) SOS1 was immunoblotted with the Abs indicated on the right.