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2 protocols using anti snf1 polyclonal antibody yk 16

1

Protein Extraction and Western Blot Analysis

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Protein extracts were prepared essentially as described previously [40 (link)]. Briefly, cells grown to exponential phase were incubated with YPD or SD medium containing 2 μg/ml tunicamycin, 4 mM DTT or 0.4 M sodium chloride, for the indicated times. Cells were transferred into test tubes, mixed 1:1 with boiled medium, submerged in the boiling water for 3 min, and harvested by centrifugation. Cells were then subjected to a mild alkali treatment-based protein extraction method [41 (link)]. Western blot analysis was performed using the immunoreaction enhancer solution Can Get Signal (Toyobo) according to the manufacturer's protocol. Anti-HA monoclonal antibody 16B12 (Covance), anti-Myc monoclonal antibody 9E10 (Santa Cruz), anti-GFP monoclonal antibody JL-8 (Clontech), anti-phospho-p38 MAPK monoclonal antibody 28B10 (Cell Signaling), anti-phospho-AMPKα monoclonal antibody 40H9 (Cell Signaling), anti-Hog1 polyclonal antibody y-215 (Santa Cruz), anti-Snf1 polyclonal antibody yk-16 (Santa Cruz), and anti-Mcm2 polyclonal antibody N-19 (Santa Cruz) were used. Detection was carried out by using a LAS-4000 (Fuji Film) with Immobilon Westren (Merck Millipore). Signal intensities were quantified by ImageQuant (GE Healthcare), and statistical analysis was performed with Excel (Microsoft).
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2

Western Blot Analysis of GFP-tagged Proteins

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Preparation of protein extracts and Western blot analysis were performed as described previously [10 (link)]. WIDE RANGE Gel Preparation Buffer(4x) for PAGE (Nakalai) was used to detect degradation of GFP-tagged proteins. Anti-GFP monoclonal antibody JL-8 (Clontech), anti-GFP antibody from mouse IgG1κ (clones 7.1 and 13.1) (Roche), anti-phospho-AMPKα monoclonal antibody 40H9 (Cell Signaling), anti-Snf1 polyclonal antibody yk-16 (Santa Cruz), anti-Myc monoclonal antibody 9E10 (Santa Cruz) and anti-Mcm2 polyclonal antibody N-19 (Santa Cruz) were used. Detection was carried out by using a LAS-4000 (Fuji Film) with Immobilon Western (Merck Millipore) or the Odyssey Imaging Systems (LI-COR Biosciences). Signal intensities were quantified by the Odyssey Imaging Systems, and statistical analysis was performed with Excel (Microsoft).
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