QUICK SEARCH:   [advanced] Author: Keyword(s):
Home     Help     Feedback     Subscriptions     Archive     Search     Table of Contents

Help viewing high resolution images Return to article

Fig. 2. PKC phosphorylates purified ezrin in T567 in vitro and in Sf9 insect cells. (A) 6xHis-tagged h-ezrin expressed in Sf9 cells and purified by Ni²⁺ columns was incubated in the absence (–) or presence of recombinant active PKC (+) (2 µg/ml) and [-³²P]ATP (5 mCi/ml, 3000 Ci/mmol). After SDS-PAGE and blotting, the membrane was exposed to X-ray film for 4 hours. After autoradiography, the same membrane was subsequently probed with anti-ezrin antibody and chemiluminescence (ezrin, 5 second exposure). (B) An identical experiment was performed in the presence of 1 mM cold ATP. The immunoblot was performed sequentially on the same membranes using anti-p-T567 ezrin, then stripped, and reprobed with monoclonal anti-ezrin. (C) As in B, identical amounts of purified 6xHis-tagged ezrin purified on Ni²⁺ columns were phosphorylated (+) or not (–) with PKC and cold ATP for 30 minutes at 30°C. The reaction was stopped by dilution and the ezrin solutions were then incubated with Sepharose beads covalently bound to phalloidin-stabilized F-actin for 4 hours. After centrifugation, the supernatants were acetone precipitated and analyzed by immunoblot with anti-ezrin antibody (sup.). The beads were extensively washed, eluted in sample buffer and the eluates analyzed by immunoblot sequentially with anti-ezrin antibody and then with anti-actin antibody on the same membranes. Positions of molecular mass standards are indicated on left: 201, 133 and 85 kDa (top to bottom). (D) Sf9 cells were infected with baculovirus expressing h-ezrin, h-PKC, or double infected with both as indicated at the left. The efficiency of infection was approximately 90% for both viruses. After 48 hours the cells were fixed and labeled for PKC (blue channel), F-actin (phalloidin, green channel) and h-ezrin (red channel). The images are X-Y confocal sections at the transnuclear level. Arrows show cells with ezrin and F-actin recruited to the cell surface. (E) Scanning electron microscopy of Sf9 cells transduced with ezrin, or PKC-expressing baculovirus, or both. (F) Extracts from parallel Sf9 cultures of cells transduced with ezrin, or PKC-expressing baculovirus, or both were purified through a Ni²⁺ column, the eluates were separated in SDS-PAGE, blotted and probed sequentially with antibodies against ezrin or T567-P ezrin. (G) Genomic DNA from noninfected, singly infected or doubly infected Sf9 cells was separated in an agarose gel and compared with a ladder of 1.6 kb to 12 kb DNA standards (left lane). A positive control for DNA laddering typical of apoptosis was performed by incubating the cells in 10 mM H₂O₂ overnight. (H) Single and double infected Sf9 cultures and a H₂O₂-positive control done as mentioned above were stained with an Apopercentage kit to detect apoptosis. Scale bars: 10 µm (D); 1 µm (E); 100 µm (H).

Return to article