Recombinant Protein Expression and Purification

DNA sequences encoding the cytoplasmic domain of BAK1, mBAK1 (L317E), SERK5 and SlBIR3 were amplified by PCR and subcloned into the pFLAG-MAC vector (Sigma-Aldrich Saint Louis, MO, USA). The primers used are listed in Table S1. Each of the resulting constructs was transformed into E. coli BL21(DE3)pLysS (Transgene, Beijing, China). The resulting recombinant proteins were purified as previously described and the autophosphorylation and their transphosphorylation activities in vitro were determined as previously described [77 (link)], using anti-FLAG (1:5000) (Transgene, Beijing, China, Cat. #HT201-01) and anti-pThr (1:2000) (CST, Danvers, MA, USA, Cat. #93815) antisera.