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. 2017 Sep 12;32(3):820–827. doi: 10.1038/leu.2017.267

Figure 2.

Figure 2

Phosphorylation of Y1196 by ABL is sufficient to elicit SOS1 RAC GEF activity. (a and b). MANT-GDP-fluorescence RAC GEF assay of purified pY1196F-SOS1. GST-RAC1 was equilibrated with 2 μM MANT-GDP. After 300 s, 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; left panels, 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 360 nm. The emission of MANT-GDP-bound to RAC was measured at 440 nm. Right panels: aliquots of phosphorylated (TCI) and phosphatases dephosphorylated (TC) SOS1 was immunoblotted with the antibodies indicated on the right.