Figure 3.

LY294002 impacts ProDH1 steady state transcript and proteins levels. Northern- and western blot analysis were performed on total RNA or proteins extracted, respectively, from 12-days-old seedlings treated with either 0.5× MS alone (control) or supplemented with 200 mM NaCl (NaCl) during 3 h or 24 h with 100 μM LY294002 (+) or DMSO (−) as described in the legend of Figure 1. (A) Total RNA (10 μg) was loaded in each lane and northern blots were probed with DNA fragments specific for AtP5CS1 and AtProDH1. Methylene blue staining of rRNAs is shown as a loading control. (B) Total proteins (20 μg) were separated on an 8% SDS-PAGE gel. Western blots were incubated with specific antibodies directed against AtP5CS and AtProDH proteins. Detection of the immunolabeled proteins was done by autoradiography using an ECL kit. Membranes were stained with Ponceau S Red as control for protein loading (Rubisco). (C) Relative expression levels of P5CS1 and ProDH1 genes measured by real-time quantitative PCR after 24 h treatment as described in (A). Results expressed as percentage compared to APT1 as a reference gene are means ± SD of 3 replicates. Letters indicate statistical differences in P5CS1 (blue letter) or ProDH1 (red letter) gene expression depending on treatment conditions (Two-Way ANOVA test, P < 0.05). The results presented in (A,C) were obtained from two independent experiments.