Figure 3: HYL1 is a short-lived protein and highly unstable but not in ubiquitin–proteasome pathway.

(a) QRT–PCR analysis of HYL1 and SERRATE transcripts in cop1-4 and cop1-6. The data were collected as average value of four biological samples with ±s.d. (n=12). (b) In vivo protein-decay assay using a translation inhibitor, cycloheximide (CHX); Left panel—wild-type seedlings with CHX, middle panel—wild-type seedlings with mock and right panel—WT/35S-HYL1-6myc transgenic seedlings with CHX. Single asterisk means endogenous HYL1; double asterisk means HYL1–6myc. (c) Concentration-dependent effects of proteolysis inhibitors on CHX-decay assay. WT seedlings were treated with CHX (0.5 mM) with or without MG132 (5, 10 and 50 μM), PIs (0.05, 0.1 and 0.2 × ), CLL (5, 10 and 50 μM), PYR-41 (5, 10 and 50 μM) or epoxomicin (0.05, 0.1 and 0.5 μM). (d) Effect of inhibitors on the stability of phyA. Seedlings were grown for 7 days under a 12-h white light/12-h darkness photoperiod and sampled 3 h after transitioning to white light. This result is an example showing the effect of UPS inhibitors on a target protein, which is regulated by ubiquitin–proteasome pathway. (e) Effects of proteolysis inhibitors on CHX-decay assay. WT seedlings were treated with CHX (0.5 mM) with or without MG132 (10 μM), PIs (0.2 × ), CLL (10 μM), PYR-41 (10 μM) or epoxomicin (0.1 μM). The levels of endogenous HYL1 were determined with α-HYL1 antibody. In all tests, the equal loading of samples was determined with α-actin antibody. (f) In vivo ubiquitination analysis of co-immunoprecipitated HYL1–6myc. HYL1–6myc was precipitated with α-myc antibody and ubiquitination pattern was determined with α-Ub antibody. Blue asterisks indicate nonspecific bands. Red asterisk indicates immunoprecipitated HYL1–6myc.