Anti lh

Leaf tissue from five-week-old transgenic Arabidopsis plants was ground under liquid nitrogen and homogenized in 5 mL/g lysis buffer [50 mM Tris-HCl (pH 7.4), 100 mM KCl, 2.5 mM MgCl₂, 0.1% NP-40, and 2× complete protease inhibitor cocktail; Roche]. After centrifugation for 15 min at 9500 × g, the clarified lysate was precleared for 20 min at 4 °C with 10 μL of bed volume protein A-agarose (30 μg protein A)/milliliter. The precleared lysates were reacted with 4 μg of anti-LH (NEB) or anti-GFP (Sigma-Aldrich)/milliliter for 1 h at 4 °C and then with 50 μL of bed volume protein A-agarose (150 μg protein A)/milliliter for 3 h at 4 °C. The precipitates were washed three times in lysis buffer and then divided for protein and RNA analyses. Nucleic acids were recovered by treatment with 3 volumes of proteinase K solution [100 mM Tris-HCl (pH 7.4), 10 mM EDTA, 150 mM NaCl, 2% SDS, and 0.2 μg/μL proteinase K] for 15 min at 65 °C, extracted with saturated phenol and phenol:chloroform, and then precipitated with ethanol. Five micrograms of RNA from the input extract or from IP fractions representing 150 mg of tissue was used for qPCR analysis. UBQ5 was used as a control.

Anti-GFP (Sigma-Aldrich, St Louis, MO, USA; F3165, 1:5000 dilution), anti-GST (Sigma-Aldrich; 1:5000 dilution), anti-LH (NEB, 1:3000 dilution) and anti-HYL1 (Agrisera, 1:1000 dilution) antibodies were used for Western blotting. The secondary antibodies used were goat-developed anti-rabbit IgG antibodies (GE Healthcare; NA931V, 1:20 000 dilution).