Figure 1.

Phosphorylation of the recombinant Flag-BRI1 (S891T) mutant at the engineered Thr-891 site. (A) Immunoblot analysis of recombinant BRI1 proteins autophosphorylated during production in E. coli, using previously described anti-pS891 antibodies and anti-pT891 antibodies developed in this study. (B) Sequence alignment of BRI1 showing two known autophosphorylation sites with significant similarity to the antigen sequence used to generate the anti-pT891 antibodies. Light and dark shading represent similar and identical amino acids, respectively. (C) Reaction of various antibodies with mBRI1, BRI1, and selected directed mutants. (D) Peptide competition of the reaction of anti-pT891 antibodies with BRI1 and the S891A and S891T mutants. Increasing amounts of the pT880 synthetic peptide (sequence: DLLQApTNGNF, where pT is phosphothreonine) were added to the primary antibodies as indicated.