Anti ha monoclonal antibody 16b12

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).

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.