Extended Data Fig. 6: Time course of ADA2 protein accumulation under stress.
(a) ADA2 protein and transcript levels under PEG stress. Upper panels: Immunoblotting analysis of ADA2 protein levels in rice seedlings treated with or without PEG during the day time, harvested at 3 h intervals. Anti-H3 was used as loading controls. Lower part: relative ADA2 transcript levels during PEG treatment. ADA2 transcript levels in rice seedlings (n = 3, mean values from 3 measurements are presented) were measured by reverse transcription quantitative polymerase chain reaction (qPCR) and normalized using Actin as the reference gene. (b) ADA2 protein and transcript levels in seedlings treated with 20% PEG6000 for 24 h with or without ActD (40 µM) treatment. Nuclear proteins were analyzed by immunoblotting with anti-ADA2 and anti-H3 antibodies. Band intensities were quantified using ImageJ software, relative to CK, and indicated below the blots. Two replicates (rep 1 &2) are shown. ADA2 transcript levels in rice seedlings (bars indicate the mean ± SE of three replicates) were measured by reverse transcription quantitative polymerase chain reaction (qPCR) and normalized using Actin as the reference gene. (c) ADA2 protein levels in rice seedling (10 days after germination) under submergence. Rice plant samples were harvested at ZT6 every 24 hours during 7 days, for acetyl-CoA and nuclear protein extraction, followed by immunoblotting with anti-ADA2 and anti-H3 acetylation antibodies. ADA2 transcript levels in rice seedlings (bars indicate the mean ± SE of three replicates) were measured by reverse transcription quantitative polymerase chain reaction (qPCR) and normalized using Actin as the reference gene.
