Fig. 4.

Monobodies inhibit SHP2 phosphorylation in cells. (A) HEK293 cells were transiently transfected with the indicated expression constructs, and total cell lysates were immunoblotted with the indicated antibodies. E76K is an activating point mutation in SHP2, and NB is a nonbinding control monobody (the Y87A mutant of HA4; ref. 19). (B) SHP2 was immunoprecipitated from HEK293 cell lysates that had been transfected with the indicated expression constructs and immunoblotted with anti-SHP2 and anti-pY (total pY) antibodies. (C) Schematic representation of the autoinhibited SHP2 conformation and activated SHP2 bound to GAB2, and the perturbation of this interaction by monobodies. The model is based on conformational changes on SHP2 activation reported previously (10, 12) and the data presented in Figs. 1E and 5A.