Phospho-Regulation of AtRGS1 in Arabidopsis

In order to identify protein phosphorylation at the C-terminal serine cluster of AtRGS1, seven-day-old rgs1-2 seedlings overexpressing TAP-tagged AtRGS1 were treated with 100 nM flg22 for 0, 3, and 15 min. Total protein was extracted as described by (15 (link)). AtRGS1 was purified using IgG-agarose beads (Sigma), and the protein A portion of TAP tag was then partially removed by TEV digestion. Phosphorylation levels at the serine cluster were determined using an anti-phospho-AtRGS1 antibody, which recognizes the pSer428, pSer435, and/or pSer436 with low avidity (10 (link)). Total AtRGS1 levels were determined using an anti-AtRGS1 antibody (9272). Both sera were produced at the University of North Carolina and are available from AgriSera AB. Detection of the phosphorylated AtRGS1 protein in vivo in the atbα mutant or in in vitro in enriched samples was not possible due to the greatly reduced abundance of AtRGS1 protein.
For the stability assay, 7-day-old seedlings were treated with the translation inhibitor cycloheximide at 200 μM for 1 h. Protein levels were compared by Ponceau S staining and AtRGS1 levels were determined by probing with anti-GFP Tag polyclonal antibody (Invitrogen #A-11122). Bands were quantified using the software ImageJ (https://imagej.net/ij/) and the RuBisCO large chain (rbcL) was used as an endogenous control of total protein levels.