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).