Fig. 2: ARF7 and ARF19 protein stability is regulated by temperature.

a Graphic depicting the ARF stability ratiometric construct that results in expression of two proteins. 16 hours after protoplast transfection, protoplasts are incubated at 22 °C or 32 °C for 4 h and fluorescence measured. b Mean ( + SE) ratio of mNeon:mScarlet signal after 4-hour treatment (n = 2652 for ARF19 at 22 °C, 1156 for ARF19 at 32 °C, 2840 for empty vector at 22 °C, and 2479 for empty vector at 32 °C), letters indicate a significance of p < 0.001, 2-way ANOVA with Tukey’s HSD test. c Graphic showing cycloheximide (CHX) treatment. d Immunoblot analysis of 5-day-old seedlings treated with the indicated temperature, time, and CHX treatment. e Immunoblot analysis of 4-day-old 35S:ARF7-HA seedlings incubated 4 h at the indicated temperature in the presence or absence of MG132. f Immunoblot analysis of four-day-old wild type (Col-0) and aff1 seedlings treated as indicated. Each immunoblot experiment was performed at least three times; additional replicates are in Supplemental Fig. 2g.