Extended Data Fig. 2. PIF7-MYC protein accumulation in response to warm temperature.

a-d Western blots of PIF7-MYC protein used for the quantification shown in Fig. 1m. The PIF7::PIF7-MYC line is in the Col-0 background. Actin levels are shown as loading control.

e, f Western blot (e) and quantification (f) of PIF7-MYC protein of an independent transgenic PIF7:: PIF7-MYC line in Col-0 background at ZT0 (dawn) and ZT12; seedlings were grown in LD at constant 17°C or with a 27 °C midday for 7 d. Protein levels were normalised to actin. Bars represent the mean, error bars indicate the SEM (n = 3). The experiment was repeated once with similar results.

g-i Western blots (g) and quantification (h) of PIF7-MYC protein as well as PIF7-MYC transcript levels (i) of PIF7::PIF7-MYC (Col-0) seedlings grown at constant 17 °C for 7 d and then either shifted to 27 °C at ZT4 (= 0 h) or kept at 17 °C for the indicated amount of time. Actin levels are shown as loading control. Protein levels were normalised to actin and expressed relative to levels at 0 h, transcript levels were normalised to PP2A and expressed relative to levels at 0 h. Data points represent the mean, error bars indicate the SEM (n = 3). The experiment was repeated once with similar results.

j, k Western blot of PIF7-MYC protein at ZT8 and ZT12 of PIF7::PIF7-MYC (Col-0) seedlings grown in LD at constant 17°C (j) or with a 27°C midday (k). Seedlings were treated with 100 μM cyclo-heximide (CHX), 50 μM MG132, a combination of the two or mock-treated at ZT4 on the day of sampling. Actin levels are shown as loading control. Two biological replicates are shown. The experiment was repeated once with similar results.

The open arrow indicates an unspecific signal. Asterisks indicate significant differences to 17 °C control treatment (Two-sided Student’s t-test, * p < 0.05, ** p < 0.01, *** p < 0.001).