Bml pw8770 0025

Total RNA was isolated from seven days old seedlings grown at the different temperatures by TRIzol method and incubated with DNase (Promega) for 30min at 37°C to remove residual genomic DNA before reverse transcription. 1 μg of total RNA was used for reverse transcription by M-MLV reverse transcriptase (Invitrogen). The transcript levels of target genes were determined by real-time PCR using specific primer sets (Supplementary Table 1) and normalized with that of PP2A. Two biological replicates were performed, with four technical replicates each. For protein analysis, seedlings were frozen in liquid nitrogen and homogenized. Total protein was extracted with urea-denaturing buffer (100 mM NaH₂PO₄, 10 mM Tris-Cl, and 8 M urea, pH 8.0, 1mM PMSF, Protease inhibitor cocktails) and the debris was removed by centrifugation. The extracted proteins were further denatured by boiling at 100°C for 5 min with 1X SDS sample buffer. The protein levels were detected by immunoblot analysis using an anti-myc antibody (Santa Cruz, c-myc (9E10) X antibody, sc-40 X, 1:10000 dilution for western blot), anti-PIF4 antibody (Agrisera, AS16 3955, 1:1000 dilution for western blot), anti-tubulin antibody (Sigma-Aldrich, anti-α-Tubulin antibody, T5168, 1:10000 dilution for western blot), anti-RPT5 (Enzo Life Sciences, BML-PW8770-0025, 1:10000 dilution for western blot).