Figure 7. Immunoblots and luciferase assays showing light regulation of CIBs protein expression.

(A) Transgenic plants expressing the 35S::Myc-CIB4 and 35S::Myc-CIB5 transgenes were grown in long day for 3 weeks, treated with blue light (Blue) for 16 hr, and transferred to dark (Dark), red light (Red, 20 µmol m−2 s−1), or far red light (FR, 5 µmol m−2 s−1) for the indicated time (Left). Alternatively, the 3-week-old plants were first treated with red light for 16 hr (Red), and transferred to blue light (Blue, 35 µmol m−2 s−1) or kept in red light (Red, 20 µmol m−2 s−1) for the indicated time. (B) Immunoblot showing the inhibition of CIB4 and CIB5 degradation in darkness by the proteasome inhibitor MG132. Plants expressing the 35S::Myc-CIB4 or 35S::Myc-CIB5 transgenes were grown in continuous white light (CW) for 3 weeks, leaves were excised and incubated with MG132 (50 µmol/L) or mock solution (0.1% DMSO) in darkness for the indicated time. (C–D) A luciferase assay showing decreased levels of LUC-CIB1, LUC-CIB2, LUC-CIB4, and LUC-CIB5 fusion proteins in the absence of blue light. Transgenic Arabidopsis seedlings expressing the indicated LUC-fusion CIB proteins were grown in continuous blue light for 7 days, and transferred to dark (C) or red light (D) for the indicated time. The luciferase activity was measured by a CCD camera (C) or by a luminometer (D). For (C)), the bioluminescence/20 seedlings were measured by a CCD camera and shown after background subtraction. For (D), the relative levels of LUC activity (REU) was calculated by the formula [LUC^Red/mg^Red]/[LUC^Blue/mg^Blue]. LUC^Blue and LUC^Red: luciferase activity of dark- or blue light-treated samples, mg^Red and mg^Blue: total proteins (mg) of dark- or blue light-treated samples.