Images used for protein quantification in Hayes et al (2019) Current Biology: Figure 3

Images used for quantification of PIF4-HA and PIF5-HA protein stability in Fig 3. Hayes et. al 2018.

35S:PIF4-HA and 35S:PIF5-HA (Ler) were germinated on ½ MS plates in white light (16:8h photoperiod) for 3 days, before being transferred to plates with or without 75mM NaCl. Plates were then grown in red light or a further 2 days. On day 5, at Zt 3, half the plates were moved to red+ far-red light. Tissues were harvested at Zt 4. 10 seedlings were homogenized and proteins were extracted in 70 µl Cracking Buffer (125mM Tris pH 7.4, 2% SDS, 10% Glycerol, 6M Urea, 5% βME) and 15 µl of the extract was run on a 10% polyacrylamide gel. Blots were probed with anti-GFP-HRP (1:1000). Membranes were developed with 50/50 ‘pico’ and ‘femto’ chemiluminescence substrate (Thermo) on a ChemiDoc (Biorad).

Experiment was performed on 4 dates (in duplicate).

R= red light

FR= red +far-red light

RN= red light +NaCl

FRN= red +far-red light +NaCl

Sample order is as follows:

01-08-18:

PIF4-HA: R1, R2, FR1, FR2, RN1, RN2, FRN1, FRN2

PIF5-HA: R1, R2, FR1, FR2, RN1, RN2, FRN1, FRN2

08-08-18:

PIF4-HA: R3, R4, FR3, FR4, RN3, RN4, FRN3, FRN4

PIF5-HA: R3, R4, FR3, FR4, RN3, RN4, FRN3, FRN4

15-08-18:

PIF4-HA: R5, FR5, RN5, FRN5, R6, FR6, RN6, FRN6

PIF5-HA: R5, FR5, RN5, FRN5, R6, FR6, RN6, FRN6

10-10-18:

PIF4-HA: R7, FR7, RN7, FRN7, R8, FR8, RN8, FRN8

PIF5-HA: R7, FR7, RN7, FRN7, R8, FR8, RN8, FRN8