Fig. 5

PtaHMGB2/3 protein abundance shows diurnal pattern. a–c PtaHMG2/3 mRNA expression profile obtained by qRT-PCR in long days conditions (LD) (a), continuous dark (DD) (b), and continuous light (LL) (c). d Immunodetection of 3xHA::PtaHMGB2/3 protein abundance obtained by western blotting. Protein extracts were obtained at ZT4 and ZT16 from wild type and 3xHA:PtaHMGB2/3 expressing Nicotiana benthamiana leaves samples grown under LD. e, f Immunodetection of 3xHA:PtaHMGB2/3 protein abundance obtained by western blotting. Nicotiana benthamiana leaves samples expressing 3xHA::PtaHMGB2/3 were collected in a 24 h time course experiment under LD conditions. Protein extracts were incubated in cell free degradation buffer during 60 min at 4 °C (e) and 30 °C (f). g Immunodetection of 3xHA:PtaHMGB2/3 protein abundance by western blot. Nicotiana benthamiana leaves samples expressing 3xHA::PtaHMGB2/3 were collected at ZT6 under LD conditions. Protein extracts were incubated in cell free degradation buffer during 30, 60 and 90 min at 30 °C or 90 min at 4 °C, with or without proteasome inhibitor MG132 50 μM. Top labelling indicates Zeitgeber times (ZT) of each protein sample. Black arrowhead shows the localization 3xHA:PtaHMGB2/3 in the blot. Red ponceau staining of the membrane is shown as loading control. All immunodetection experiments were performed twice. A representative result is shown in this figure