Figure S2: Identification of new protein interactors with SKN-1/Nrf. A. Dominant mutations that activate SKN-1 were isolated in an EMS mutagenesis screen of a C. elegans strain harboring the SKN-1 transcriptional reporter gst- 4p::gfp. F1 generation worms with GFP expression were isolated for further characterization. B. F2 generation animals were singled and F3 progeny were analyzed for 100% penetrance of the activated GFP phenotype. These homozygous animals were then mated back to the unmutagenized parental strain. Successful matings that yielded 100% F1 progeny expressing the activated GFP phenotype were confirmed as dominant mutants. C. Two dominant mutations activate the
gst-4p::gfp reporter of SKN-1 activity in C. elegans intestinal and hypodermal tissues. D. Bioinformatic (Expasy Prosite) identification of potential phosphorylation sites (light line), Casein kinase 2 recognition site (dark line), and potential phosphorylation sites (*). E. yeast-2- hybrid screen identifies PGAM-5, MXL-3, D1025.1, K10D3.4, HMG-1.1 as protein-protein interactors with the new SKN-1 50-mer domain. F. SKN-1 interacting proteins PGAM-5, K10D3.4, and MXL-3 were tested for binding, using WDR-23 as bait. G. Truncated versions of MXL-3 were tested for binding with SKN-1. H. 35S-methionine labeled SKN-1A-HAtag (lane 1), SKN-1B-HAtag (lane 2), MXL-3 (lane 3), PGAM-5 (lane 4), positive control WDR-23 (lane 5), and negative control Luciferase (Lane 6) were expressed using a Promega TNT coupled in vitro transcription and translation System. MXL-3 (green circles), PGAM-5 (red circles) and WDR-23 (blue circles) could bind to SKN-1A:HA (lane 7, 8, and 9) and SKN-1B:HA (lane 11, 12, and 13). Neither SKN-1A:HA or SKN- 1B:HA could bind to luciferase (lane 10 and 14). Top panel exposure time 6 hours, bottom panel exposure time 24 hours. Worms harboring extrachromosomal arrays of
mxl-3::gfp (I) or
pgam-5::gfp (J) were mounted on agar pads and imaged for GFP fluorescence. Arrows indicated expression in unidentified neuronal and pharyngeal cells.