Anti phospho ampkα monoclonal antibody 40h9

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).

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).