Cold-induced degradation of FAX1 by RBL11 in Arabidopsis. A) Scheme of experimental setup. B) Immunoblot analysis via HA antibody of soluble and insoluble (membrane) protein extracts from Wt and transgenic plants expressing RBL11 tagged with a HA epitope (∼32 kDa) under the control of a 35S promoter after induction with DEX for 48 h at 21 °C. C) Immunoblot detection via HA antibody of soluble and insoluble (membrane) protein extracts from Wt and transgenic plants expressing RBL11 tagged with a HA epitope (∼67 kDa) under the control of a 35S promoter after induction with DEX for 48 h at 4 °C. Note that the size of the protein (∼67 kDa) comes from the presence of an additional biotinylase (∼35 kDa), which was not relevant in this experiment. D) Immunoblot of insoluble protein extracts from Wt and transgenic plants overexpressing RBL11 under the control of a 35S promoter after induction with DEX for 48 h at 21 °C or 4 °C with a FAX1 antibody. Please note that the extraction of whole membranes enriched from cold-treated leaves generally resulted in a higher abundance of FAX1 and is not comparable with results from leaves treated at 21 °C. Ponceau staining in B), C), and D) is representing equal loading of protein samples with 18 µg per lane.