2.11. SUnSET assays

SUnSET assays were carried out using published protocols with minor modifications (Van Hoewyk, 2016). Nine‐day‐old maize seedlings were treated with water (mock) or 5 µg/ml TM for 6 and 12 hr followed by treatment with 50 µM puromycin solution (Sigma‐Aldrich P8833‐25MG) or water (no puromycin negative control) for 30 min. Seedlings were rinsed with water and 200–300 mg of roots were ground with mortar and pestle in liquid nitrogen followed by addition of protein extraction buffer (20 mM Tris‐HCl (pH 7.5), 50 mM NaCl, 1 mM EDTA (pH 8), 0.1% (v/v) Triton X‐100, 10% (v/v) glycerol, 2 mM NaF, 1 mM PMSF, 5 mM DTT, 1 tablet of Mini Complete protease inhibitor cocktail (Roche 4693124001)). The crude lysate was vortexed, centrifuged at 21k g for 3 min at 4°C, and the supernatant was centrifuged again at 21k g for 5 min at 4°C followed by quantification using a Qubit protein assay kit (Invitrogen {"type":"entrez-protein","attrs":{"text":"Q33212","term_id":"75281053","term_text":"Q33212"}}Q33212). About 20 µg of total protein was mixed with equal volume of 2x Laemmli buffer (with β‐mercaptoethanol; BioRad), incubated in boiling water for 5 min and loaded in 4%–15% Mini‐protean TGX stain‐free protein gel (BioRad). Electrophoresis was carried out in 10x Tris/glycine/SDS buffer (BioRad) at 100 V for 100 min followed by imaging for total protein as a loading control. Subsequently, proteins were transferred from gel to a PVDF membrane using Invitrogen iBlot2 instrument, the membrane was blocked with 5% nonfat milk at room temperature (RT) for 1 hr, washed with 1x TBST (Tris‐buffered saline with Tween 20) at room temperature for 5 min, incubated with anti‐puromycin primary antibody (DSHB, University of Iowa, PMY‐2A4) in 0.5% nonfat milk (antibody final concentration was 0.48 µg/ml) overnight at 4°C, washed three times with 1x TBST for 5 min, incubated with secondary antibody (anti‐mouse IgG from sheep‐conjugated with HRP) in 0.5% nonfat milk (3:10,000 dilution) at room temperature for 1 hr and washed three times with 1x TBST for 5 min. Images were developed using a SuperSignal West Pico Plus chemiluminescent kit (Thermo Scientific) according to the manufacturer's protocol.