Supersignal west atto chemiluminescent substrate

Cells were lysed in Pierce RIPA buffer (Thermo Fisher Scientific) supplemented with 1% HALT protease and phosphatase inhibitor cocktail (Thermo Fisher Scientific). Extracted protein samples were normalized using the Pierce BCA Protein Assay Kit (Thermo Fisher Scientific), fractionated on Bolt 4%–12% Bis-Tris gradient gels (Invitrogen), and transferred to 0.2-μm PVDF membranes (GE Healthcare). Primary antibodies and HRP-conjugated secondary antibodies are shown in Supplemental Table 5. SuperSignal West Femto or SuperSignal West Atto chemiluminescent substrate (Thermo Fisher Scientific) was used for imaging. Fiji (ImageJ) was used to analyze band intensity.

Protein extracts were resolved by SDS-polyacrylamide gel electrophoresis on Bolt NuPAGE 4–12% Bis-Tris Plus gels (Invitrogen, ThermoFisher Scientific; #NW04122BOX) using Bolt NuPAGE MOPS SDS running buffer and transferred to PVDF blotting membranes (Amersham Hybond) with Trans-Blot SD Semi-Dry Transfer Cell. The following antibodies were used: Phospho-IRF-3 (Ser396) (4D4G) Rabbit mAb (Cell Signaling, #4947); IRF-3 (D614C) Rabbit mAb (Cell Signaling, #11904); EiF4G (C45A4) Rabbit mAb (Cell Signaling, #2469) and β-Actin (8H10D10) antibody (Cell Signaling, #3700). HRP-coupled anti-mouse (NA9310V, GE Healthcare) and anti-rabbit IgG, HRP-linked antibody (Cell Signaling, #7074) were used as secondary antibodies. Peroxidase activity was visualized with SuperSignal West Pico PLUS chemiluminescent substrate (#A34580, ThermoFisher Scientific) or SuperSignal West Atto chemiluminescent substrate (#A38556, ThermoFisher Scientific) with the Invitrogen iBright CL1500 Imaging System.

Total protein extracts were made from 50 seedlings for each shoot or root sample using 50 μL urea extraction buffer, consisting of 8 M urea, 0.35 M Tris-Cl pH 7.5, and 1× protease inhibitor cocktail. After boiling with 6× SDS sample buffer, samples were centrifuged at 20,200 × g for 15 min and loaded into SDS-PAGE gels. Separated proteins were transferred onto PVDF membrane (Millipore) and then immunoblotted using anti-GFP (Abcam, ab290, dilution 1:5000), anti-PIF4 (Agrisera, AS16 3955, dilution 1:2000), or anti-HY5 (Abiocode, R1245-2, dilution 1:3000) antibodies. For the secondary antibodies, anti-mouse (Abcam, ab131368, dilution 1:5000), anti-rabbit (KPL, 95059-086, dilution 1:50,000), or anti-goat (Agrisera, AS09 605, dilution 1:5000) were used. Coomassie blue staining (CBB), anti-Tubulin (Sigma-Aldrich, T5168, dilution 1:5000), or anti-RPT5 (Enzo Life Sciences, BML-PW8770-0025 dilution 1:5000), was used for loading control. SuperSignal West Atto Chemiluminescent substrate (ThermoFisher Scientific, A38556) was used for visualizing secondary HRP bound antibodies.