Yeast protein extraction reagent

Cell extract was prepared with Yeast Protein Extraction Reagent (Thermo Scientific, Pierce, Rockford, USA) according to the instructions provided by the manufacturer. The total protein concentration in cell extracts was determined using the Bradford method [34 (link)] with bovine serum albumin (BSA) as standard. SADH activity measurements were performed as described previously [35 (link)]. The activity is based on measuring the oxidation of NADH at 340 nm with an Ultrospec 2100 pro spectrophotometer (GE Healthcare Life Sciences, Sweden). The data were collected with the software program SWIFTII (Amersham Biosciences, Sweden). Cell extracts were diluted until the decrease in absorbance was linear for 5 min, at which point the activity could be calculated from the slope. One unit of activity corresponds to 1 µmol NADH consumed per minute at 25 °C. The assay contained sodium phosphate buffer (50 mM, pH 7), acetophenone (10 mM), NADH (0.2 mM), and cell extract (1-30 mg/l total protein).

Genomic DNA from bacteria and yeast strains was extracted with PCI [phenol/chloroform/isoamyl-alcohol (25:24:1)] as previously described [16 ]. DNA extraction from agarose gels and purification of PCR products were performed using Wizard SV Gel and PCR Clean Up System (Promega). Polymerase chain reaction (PCR) was performed with Phusion DNA polymerase (Thermo Fischer Scientific) for construction of the vectors, and with GoTaq polymerase (Promega) for diagnostic purposes. Sanger sequencing was performed in a 3500 Genetic Analyzer (Applied Biosystems) using “Big Dye Terminator v3.1 Cycle Sequencing Kit” (Applied Biosystems) according to the manufacturer’s instructions. DNA was transformed into yeast cells using a standard lithium acetate method [16 ]. Total protein extraction from yeast strains was performed using Yeast Protein Extraction Reagent (Thermo Fischer Scientific) following the manufacturer’s instructions.

For the assay in yeast, the vectors of LexA fused wild-type and mutated UVR8 were transformed into the yeast strain EGY48 (Clontech). Total proteins were extracted from transformants in Yeast Protein Extraction Reagent (Thermo), 1 mM phenylmethylsulfonyl fluoride (PMSF), and 1× complete protease inhibitor cocktail (Roche), and then kept on ice under −UV-B (3 µmol·m⁻²·s⁻¹ of white light) or +UV-B (3 µmol·m⁻²·s⁻¹ of white light and 1.5 µmol·m⁻²·s⁻¹ of UV-B) for 20 min. Added with 4× loading buffer containing 250 mM Tris-HCl (pH 6.8), 2% SDS, 20% β-mercaptoethanol, 40% glycerol, and 0.5% bromophenol blue, the samples were subjected to immunoblot analysis without boiling.
The assay in plants was performed as previously described [17] (link). Total proteins were extracted from 4-day-old Arabidopsis seedlings grown under −UV-B or +UV-B in protein extraction buffer containing 50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1 mM EDTA, 10% glycerol, 0.1% Tween 20, 1 mM phenylmethylsulfonyl fluoride (PMSF), and 1× complete protease inhibitor cocktail (Roche). Then the cell extracts were kept on ice under exactly the same condition (−UV-B or +UV-B) as where the seedlings were grown for 30 min. Added with 4× loading buffer containing 250 mM Tris-HCl (pH 6.8), 2% SDS, 20% β-mercaptoethanol, 40% glycerol, and 0.5% bromophenol blue, the samples were subjected to immunoblot analysis without boiling.