Journal: Scientific Reports

Article Title: Expression control of the AMPK regulatory subunit and its functional significance in yeast ER stress response

doi: 10.1038/srep46713

Figure Lengend Snippet: ( a , b ) Splicing of HAC1 mRNA after DTT treatment. Wild-type (WT) and indicated mutant strains were grown at 25 °C until exponential phase and treated with 4 mM dithiothreitol (DTT) for the indicated time. Total RNAs prepared from each strain were subjected to RT-PCR of HAC1 . Positions of unspliced HAC1 (HAC1 u ) and spliced HAC1 (HAC1 s ) are indicated. The mean of HAC1 s /( HAC1 u + HAC1 s ) at 3 hours after DTT addition with SEM (n = 4) is shown in ( b ). * P < 0.05 and ** P < 0.01 as determined by Tukey’s test. Original data are presented in . ( c ) Hog1 activation after DTT treatment. Wild-type (WT) and indicated mutant strains were grown at 25 °C until exponential phase and treated with 4 mM dithiothreitol (DTT) for 3 hours. Extracts prepared from each cell were immunoblotted with anti-phospho-p38 (P-Hog1) and anti-Hog1 antibodies. Original data are presented in . ( d ) Loss of Gal83 has the strongest influence on ER stress sensitivity. Wild-type (WT) and indicated mutant strains were spotted onto YPD medium lacking or containing 0.5 μg/ml tunicamycin (TM) and incubated at 25 °C.

Article Snippet: Anti-GFP monoclonal antibody JL-8 (Clontech), anti-phospho-p38 MAPK monoclonal antibody D3F9 (Cell Signaling), anti-Hog1 polyclonal antibody y-215 (Santa Cruz), anti-phospho-p44/42 MAPK polyclonal antibody (Cell Signaling), anti-Mpk1 polyclonal antibody yN-19 (Santa Cruz), and anti-Mcm2 polyclonal antibody N-19 (Santa Cruz) were used.

Techniques: Mutagenesis, Reverse Transcription Polymerase Chain Reaction, Activation Assay, Incubation