GST pulldown of transiently transfected IRS-2 and mutants.

<p><i>A</i> and <i>B</i>. GFP-IRS2, GFP-IRS2-S573A and GFP-IRS2-S556A/S573A were expressed transiently in HEK293 cells and cells were stimulated with 50 ng/ml IGF-1 for 30 min or after preincubation with 100 nM wortmannin for 30 min. Lysates were incubated with 2 µg GST-14-3-3β or ε for 2 hours. Samples were separated on 7.5% SDS gels and membranes were probed with GFP and GST antibodies.