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. 2019 Oct 8;116(43):21925–21935. doi: 10.1073/pnas.1904818116

Fig. 6.

Fig. 6.

Phosphorylation of MYBS2 at Ser53 is necessary for interactions with 14-3-3 (GF14) proteins in the cytoplasm. (A) Consensus 14-3-3 binding motif (underlined) in MYBS2. Potential phosphorylation residues Ser51-53 (red font) in MYBS2 were identified by LC/MA/MS (SI Appendix, Fig. S6A). (B) Rice embryo calli were cotransfected with Ubi:MYBS2 (point mutated)-mCherry and Ubi:GFP-GF14D constructs and incubated in +S medium for 24 h. Total proteins were extracted and GFP-GF14D was isolated using GFP trap. Anti-GFP and anti-RFP antibodies were then used to detect coprecipitated GF14 and MYBS2 proteins, respectively, by means of immunoblotting. (C–F) Barley aleurones were transfected with point-mutated MYBS2 alone or with 14-3-3 protein (GF14D), then incubated in +S (C and D) or −S (E and F) medium for 24 h, before being examined under confocal microscopy. (Scale bars, 50 µm.) “N” and “C” indicate higher GFP or mCherry signals in the nucleus and cytoplasm, respectively, whereas “n” represents lower signals in the nucleus. (C and E) Barley aleurones were transfected with Ubi:MYBS2(S51A)-GFP, Ubi:MYBS2(S52A)-GFP or Ubi:MYBS2(S53A)-GFP. (D and F) Barely aleurones were cotransfected with Ubi:MYBS2(mutant)-mCherry and Ubi:GFP-GF14D.