Figure 8.
HEC2 Is Partially Degraded in the Dark, and Light Stabilizes HEC2-GFP Posttranslationally.
(A) HEC2-GFP protein is accumulated upon white light exposure. Four-day-old dark-grown seedlings of HEC2-GFP transgenic seedlings were either kept in darkness or exposed to 75 μmol m−2 s−1 white light for the time indicated before samples harvested for protein extraction. Around 30 μg total proteins per sample were separated on 8% polyacrylamide gel and transferred onto PVDF membrane for immunoblot analysis. Anti-GFP and anti-RPT5 antibodies were used for detecting HEC2-GFP level or RPT5 level as a control.
(B) HEC2-GFP is degraded in darkness. Seven-day-old white light-grown seedlings of HEC2-GFP transgenic lines were kept under the same white light condition or in the dark for the time indicated before protein extraction. The immunoblot process was done as described in (A).
(C) and (D) Quantification of HEC2-GFP level in the conditions indicated in (A) and (B). Three biological repeats were performed. The band intensities were measured with ImageJ. The HEC2-GFP protein level in each sample has been normalized using the RPT5 level.
(E) HEC2-GFP protein accumulated upon exposure to all three monochromatic lights. Four-day-old dark-grown seedlings of HEC2-GFP transgenic lines were kept either in darkness or exposed to R light (Rc; 20 μmol m−2 s−1), FR light (FRc; 10 μmol m−2 s−1), or blue light (BL; 20 μmol m−2 s−1) for 6 h. The immunoblot process was done similar to that in (A).
(F) Quantification of HEC2-GFP level in the conditions indicated in (E). Three biological repeats were performed. The band intensities were measured with ImageJ. The HEC2-GFP protein level in each sample has been normalized using the RPT5 level.
(G) HEC2-GFP is stabilized in darkness by the 26S proteasome inhibitor Bortezomib. Four-day-old dark-grown seedlings of HEC2-GFP transgenic lines were treated with 40 μmol Bortezomib for either 3 or 6 h in the dark. Dark samples without treatment before and after 6 h were used as control to indicate the HEC2-GPF protein level in darkness. Four-day-old dark-grown seedlings plus 6 h white light treatment (75 μmol m−2 s−1) were used to present HEC2-GPF under light condition. The immunoblot was done as described in (A).
(H) Quantification of HEC2-GFP level in the conditions indicated in (G). Three biological repeats were performed. The band intensities were measured with ImageJ. The HEC2-GFP protein level in each sample has been normalized using the RPT5 level.