As09647

Arabidopsis proteins were extracted from 10-day-old seedlings using NEB buffer (20 mM HEPES, pH 7.5, containing 40 mM KCl, 1 mM EDTA, 1 mM PMSF, and 1× protease inhibitor cocktail [Roche, Basel, Switzerland]) after centrifugation at 12,000 × g, 4°C, for 20 min. Co-IP was performed as described previously [75 (link),76 (link)], with minor modifications. In total, 50 μl of the supernatant was collected as input. The rest of the supernatant was used for immunoprecipitation using 10 μl of GFP-Trap agarose beads (ChromoTek, Martinsried, Germany). After incubation for 2 h at 4°C, the beads were washed five times in wash buffer (20 mM HEPES, pH 7.5, 40 mM KCl, and 0.1% Triton X-100). An appropriate amount of 2× SDS sample buffer was then added to the beads, which were boiled for 10 min at 100°C, and then subjected to 10% SDS-PAGE for immunoblot analysis using antibodies against GFP (G1544-100UG; Sigma-Aldrich) and GSNOR (AS09647; Agrisera, Vännäs, Sweden). All experiments were repeated independently three times; representative results from a single experiment are shown.

Seven-day-old seedlings (around 30 plants) grown on ½ MS medium or ½ MS medium supplemented with 300 µM Fe were harvested, weighed and ground in liquid nitrogen to fine powder. In total 5 volumes (5 µl. mg⁻¹) of 2 × NuPAGE lithium dodecyl sulfate (LDS) sample buffer (Thermo-Fischer) with Tris(2-carboxyethyl) phosphine Hydrochloride (TCEP) was added to the samples and then boiled for 5 min. After centrifugation at 15,000 × g at 4 °C for 15 min, 15 μL of supernatant were loaded, separated in a NuPAGE 4–12% Bis-Tris protein gel (Thermo-Fischer), followed by a wet transfer to nitrocellulose membrane (Bio-Rad). The GSNOR protein was detected by probing the membrane with anti-GSNOR antibody (AS09 647, Agrisera) at a dilution of 1:2000. Loading was controlled by using a rabbit anti-histone H3 antibody (9715 S, Cell Signaling Technology) at a dilution of 1:2000. Signal was detected using the SuperSignal™ West Pico PLUS Chemiluminescent Substrate (Thermo-Fischer). At least two independent biological replicates (the samples from two different experiments) were performed for the western blot. The representative images from one experiment were presented.