Figure 2. Cbl binds directly to phosphorylated Tyr⁵⁶⁸ and Tyr⁹³⁶ and the specificity is determined by the amino acids at the +3 positions to the tyrosine residues respectively.

(A) Phosphopeptides pY568 and pY936 (sequences in the Experimental section) immobilized to Affigel 10 beads were incubated (3 h at 4 °C) with the indicated purified GST fusion proteins in the presence of either unphosphorylated or phosphorylated peptides in solution as indicated. Samples were analysed by SDS/PAGE and Western blotting, and membranes were probed with an anti-GST antibody. (B) Pulldown experiment performed as described above with competition by soluble peptides (100 μM) as indicated.