Figure 3. Immunoblot assays showing PPK-dependent catalysis of the blue light-dependent phosphorylation of CRY2.

(a) HEK293T cells co-expressing CMV::Myc-CRY2 and individual CMV::Flag-GFP-PPK (PPK1 to PPK4) were grown in the dark without blue light treatment (Blue −) or exposed to blue light (Blue +, 30 μmol m⁻² s⁻¹) for 2 h. Lysates were treated without (−λ-PPase) or with λ-PPase (+λ-PPase) for 30 min, and analysed by immunoblots probed with the anti-CRY2 (top two panels) or anti-Flag antibody (α-Flag) respectively. (b) HEK293T cells co-expressing CMV::Myc-CRY2 and CMV::Flag-GFP-PPK1 were exposed to green (30 μmol m⁻² s⁻¹), blue (30 μmol m⁻² s⁻¹), red (30 μmol m⁻² s⁻¹) or far-red light (5 μmol m⁻² s⁻¹) for 60 min, and analysed by immunoblots as in a. (c–e) Same as (b), except that CMV::Myc-CRY2 was co-transfected with CMV::Flag-GFP-PPK2 (c), CMV::Flag-GFP-PPK3 (d) or CMV::Flag-GFP-PPK4 (e).