Journal: PLoS ONE
Article Title: The Dynamic of the Splicing of bZIP60 and the Proteins Encoded by the Spliced and Unspliced mRNAs Reveals Some Unique Features during the Activation of UPR in Arabidopsis thaliana
Figure Lengend Snippet: Inhibition of the proteasome activity leads to the detection of GFP-bZIP60u on the ER. Seedlings (7-days-old) of transgenic Arabidopsis plants harbouring the construct 60pro:GFP-bZIP60 were pretreated with DMSO (0.2% v/v) or MG132 (100 mM) during 18 hours. Then seedlings were treated with DMSO (0.2% v/v) (A-D), DMSO (0.2% v/v) plus DTT (2 mM) (E-H), MG132 (100 μM) (I-L) or MG132 (100 μM) plus DTT (2 mM) (M-P) during 3 hours. All treatments were performed in 0.5x MS phytagel plates supplemented with the chemicals mentioned above at the indicated concentrations. After treatment, seedlings were stained with ER-Tracker Blue-White DPX (1 μM) during 45 minutes, rinsed and stained with FM 4–64 (5 μM) during additional 5 minutes at room temperature. Finally, roots were analyzed by confocal microscopy. Scale bar equals 5 μm. Images are representative of three independent experiments. (Q) Immunoblot analysis of total protein extracts obtained from bzip60 / 60pro:GFP-bZIP60 transgenic Arabidopsis seedlings (7-days-old) treated with DMSO (0.2% v/v), MG132 (100 μM) or MG132 (100 μM) plus DTT (2 mM) as mentioned above, using a polyclonal antibody against GFP. bzip60 mutant seedlings were used as control. Anti-PEPC antibody was used as loading control. Data are representative of three independent experiments. (R) CE-LIF analysis of RT-PCR products obtained from total RNA extracted from roots of bzip60 mutant or bzip60 / 60pro:GFP-bZIP60 transgenic Arabidopsis plants (7-days-old) treated with DMSO (0.2% v/v), DMSO (0.2% v/v) plus DTT (2 mM), MG132 (100 μM) or MG132 (100 μM) plus DTT (2 mM) as described above. Spherograms peaks, corresponding to the unspliced form of bZIP60 (U) and spliced form (S), are depicted. Asterisks indicate the detection of a third peak only present in DTT treated samples. Data are representative of three independent experiments.
Article Snippet: For subcellular imaging, seedlings of 7 days-old treated with DMSO, DMSO plus DTT, MG132 or MG132 plus DTT were stained with ER-Tracker Blue-White DPX dye (Molecular Probes, Life Technologies) diluted on liquid MS media during 45 minutes, rinsed on liquid MS media and stained with FM 4–64 dye (Molecular Probes, Life Technologies) during 5 minutes; at room temperature.
Techniques: Inhibition, Activity Assay, Transgenic Assay, Construct, Mass Spectrometry, Staining, Confocal Microscopy, Mutagenesis, Reverse Transcription Polymerase Chain Reaction