Nupage gel

In vitro kinase assay was done as described previously (Alassimone et al, 2016) with small modifications. Purified 250 ng GST‐SGN3 kinase domain was incubated with 250 ng TF‐SGN1 or TF proteins in reaction buffer (50 mM HEPES‐KOH pH7.5, 1 mM MnCl₂. 1 mM DTT, 1 mM ATP, 185,000 Bq [γ‐³²P] ATP) at 30°C for 30 min. 250 ng TF‐SGN1 and 1 μg GST or 500 ng GST‐N‐terminal cytoplasmic domains of RBOHD or F were treated in the same way as above. The reaction was stopped by adding 4 × LDS sample buffer (Invitrogen) and heating at 75°C for 10 min. The samples were separated on 4–12% gradient Nu‐PAGE gels or 10% Nu‐PAGE gels (Invitrogen). After drying the gels, signal was detected using Typhoon FLA7000 (GE Healthcare).

To analyze protein composition of the pelleted virions, the pellets were suspended in 50 μl of 1X non-reducing SDS sample buffer (BioRad, Richmond, CA), and then 10 μl of the RA27/3 preparation and 1 μl RV-Dz preparation were separated by 4–12% NuPage gels (Invitrogen, Carlsbad, CA). Different running buffers were used to separate proteins in conformations optimal for detection with each antibody, non-reducing (E1 MAb) or reducing (E2, C MAbs). To analyze viral proteins in infected cells, the cell monolayers were washed 3 times with ice-cold PBS and then proteins were extracted with RIPA buffer (Thermo Scientific, Rockford, IL) supplemented with Halt protease cocktail (Thermo Scientific, Rockford, IL). Protein concentration in the extracts was determined by BCA assay (Thermo Scientific, Rockford, IL). Equal amounts of total protein (10 μg/lane) for each sample were separated by 4–12% NuPage gels (Invitrogen, Carlsbad, CA) using MOPS running buffer and then transferred onto nitrocellulose membrane. SNAP i.d. Protein Detection System (Millipore) was used to process western blots as described earlier [18 (link)]. Blots were developed with the ECL-plus detection reagents (GE Healthcare, Piscataway, NJ) and exposed to X-ray film. The intensity of capsid band was quantified by densitometry with Carestream Molecular Imaging Software (Carestream Health Inc., New Haven, CT).

To identify the phospho-sites in RGS1, WT protoplasts expressing RGS1CT-FLAG was treated with 1 μM flg22 for 5 min, lysed in protein IP buffer (50 mM HEPES [pH 7.5], 150 mM KCl, 1 mM EDTA, 0.5% Trition-X 100, 1 mM DTT, proteinase inhibitor cocktail), and the RGS1CT-FLAG protein was purified using anti-FLAG M2 agarose (Sigma). The immunoprecipitates were separated in 10% NuPAGE gel (Invitrogen) and subject to LC-MS/MS analysis as previously described.^{23 (link)}To identify BIK1- and PBL1-phosphorylated sites in RGS1, the GST-tagged BIK1, PBL1, or BIK1^K105E was co-expressed with HIS-tagged RGS1CT in E. coli and purified using Ni-NTA agarose beads. RGS1CT-HIS protein was separated in 10% NuPAGE gel (Invitrogen) and subjected to LC-MS/MS analysis.
For in vitro phosphorylation of RGS1 isolated from protoplasts, Arabidopsis protoplasts were transfected with 100 μg RGS1CT-FLAG plasmid. RGS1CT-FLAG was affinity-purified, incubated with 200 ng HIS-BIK1 in the kinase reaction buffer (25 mM Tris-HCl, 10 mM MgCl 2, 1 mM DTT, 100 mM ATP, pH 7.5) for 30 min at 30 °C, and phosphorylation of RGS1 (band-shift) was detected by immunoblot analyses.