Phot1 containing a modified gatekeeper residue (T740G) can accommodate <t>N</t> 6 <t>-benzyl-ATPγS</t> and undergo thiophosphorylation in vitro . A , immunoblot of a kinase assay containing cell-free expressed wildtype phot1, phot1-D806N, or phot1-T740G in the presence of N 6 -benzyl-ATPγS. Reactions were carried out in the absence ( D ) or presence of 20 s of white light ( L ), and thiophosphorylation was detected using anti-thiophosphoester antibody (α -thioP ). An immunoblot analysis of phot1 protein levels using anti-HA antibody is shown below. B , phot1-T740G thiophosphorylation in the presence of the kinase inhibitor 1-NM-PP1 or DMSO as a control. Ponceau staining of cell-free expression reactions is shown below to indicate equal protein loading.
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