Extended Data Fig. 8: VIL1 protein is stabilized by light and VIL1 enrichment is dependent on phyB.
a and b, Diurnally regulated VIL1-myc protein levels (a) and VIL1 mRNA levels (b). a, The seedlings of pVIL1:VIL1-myc/vil1-1 transgenic plants grown at 22 °C in SD for 7 days were collected at eight different time points (ZT0 to ZT20). The VIL1-myc protein levels were detected by anti-myc antibody. Endogenous tubulin levels were detected as controls by anti-tubulin antibody. b, VIL1 mRNA levels in Col-0 seedlings grown at 22 °C in SD for 7 days. Samples were collected at eight different time points (ZT0 to ZT20). The expression levels are normalized to that of PP2A. Error bars: ± s.d., n = 2, biological replicates (each biological replicate is an average value of four technical replicates). c, VIL1-myc protein levels were stabilized in light. Four-day-old dark grown seedlings were either kept in the dark or treated with continuous red-light (10.7 µmol m-2 s-1) for the period indicated in the panel. Anti-myc and anti-RPT5 antibodies were used to detect VIL1-myc and RPT5 (control), respectively. d, Unchanged VIL1-myc protein levels in phyB-9 mutants. Proteins were extracted in the seedlings grown for 7 days at 22 °C in SD at ZT6. In a, c and d, all experiments were repeated at least two times independently with similar results. e, FLC locus with ChIP-qPCR amplicons (P1 and P2). f, Similar enrichments of VIL1 on FLC locus between at ZT0 and at ZT6. Error bars: ± s.d., n = 3, biological replicates. g, ChIP assay showing that relative enrichments of VIL1-myc on ATHB2 are reduced in phyB-9 at ZT6. Error bars: ± s.d. n = 3, biological replicates. f and g, The ChIP assay used anti-myc antibody on seven days old seedlings grown at 22 °C in SD. The immunoprecipitated DNA relative to input was further normalized to that of 5 S rDNA. In f and g, the asterisks indicate statistical difference in a two-tailed student t-test (***P < 0.0005, *P < 0.05), n.s. indicates not significant.
