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2 protocols using anti ha monoclonal antibody 16b12

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

Parasite Membrane Protein Extraction and Detection

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Saponin parasite pellets were lysed by sonication in phosphate buffered saline with protease inhibitors (10 µM E–64, 10 µM pepstatin A and 1 mM 4-(2-aminoethyl)benzenesulfonyl fluoride). Lysates were centrifuged at 16,100× g for 10 minutes at 4°C to remove hemozoin. Samples from parasites expressing PfSortilin-HA were further centrifuged at 100,000× g for 1 hour at 4°C to isolate the membrane fraction, which was solubilized in reducing, SDS-containing Laemmli buffer. Proteins were resolved on polyacrylamide gels and blotted to nitrocellulose. Antibodies used were anti-RFP (Rockland), 0.3 µg/mL; anti-HA monoclonal antibody 16B12 (Covance), 1 µg/mL; anti-plasmepsin V [83] (link), 1∶400; and affinity purified rat anti-PfGDPD [86] (link), 1∶5000. Signal was detected by chemiluminescence using horseradish peroxidase-conjugated anti-rabbit or anti-mouse secondary antibodies. Blots were developed using ECL Plus (GE Biosciences) and imaged on a Storm 840 imager (GE Biosciences) or on film.
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