Figure 5: Immunoblot analyses of the substrate specificity of PPKs.
From: Molecular basis for blue light-dependent phosphorylation of Arabidopsis cryptochrome 2
(a-c) HEK293T cells expressing CMV::Myc-CRY2 (a,b) or chromophore-deficient CMV::Myc-CRY2D387A (c) in the absence (a) or presence of the co-expressed CMV::Flag-GFP-PPK1 (b,c) were exposed to blue light (30 μmol m−2 s−1) for the indicated time. CRY2 and PPK1 were detected by the anti-CRY2 or anti-Flag antibodies, respectively. (d) HEK293T cells expressing CMV::Myc-hCRY2 or CMV::Myc-AtCRY2 in the absence (PPK1−) or presence of CMV::Flag-GFP-PPK1 (PPK1+) were exposed to blue light (30 μmol m−2 s−1) for 2 h. Lysates were treated with (λ-PPase +) or without λ-PPase (λ-PPase −), and analysed by immunoblots probed with the anti-Myc (α-Myc) or anti-Flag (PPK1) antibody, respectively. (e) HEK293T cells co-expressing CRY2 and PPK1 or indicated kinases are analysed as in a. (f) HEK293T cells co-expressing CMV::Myc-CRY2 and CMV::Flag-PPK1 in the presence of CMV::GFP-BIC1, CMV::GFP-GFP-BIC2, or CMV::GFP were kept in the dark (Blue −) or exposed to blue light (30 μmol m−2 s−1, Blue +) for 1 h, and analysed by immunoblots probed with anti-CRY2 (CRY2), anti-Flag (PPK1) or anti-GFP (BIC1, BIC2 or GFP) antibody, respectively.
