One million cells were lysed in NuPAGE LDS Sample Buffer (Invitrogen, Carlsbad, CA) followed by sonication. Samples were heated at 70°C for 10 min, separated by SDS–polyacrylamide gel electrophoresis, and then transferred onto nitrocellulose membranes (Invitrogen, Carlsbad, CA). The membranes were washed in Tris-buffered saline (TBST) for 3 min and then blocked for 30 min at room temperature with 5% milk. The blots were incubated with a mouse anti-cofilin antibody (1:1000 dilution; BD Biosciences, San Jose, CA) and a rabbit anti–phospho-cofilin (serine 3) antibody (1:500 dilution; Cell Signaling) diluted in 3% milk-TBST and rocked overnight at 4°C. The blots were washed three times for 15 min and then incubated with DyLight 680 goat anti-mouse and DyLight 800 goat anti-rabbit antibodies (KPL, Gaithersburg, MD) (1:5000 diluted in blocking buffer) for 1 hour at 4°C. The blots were washed three times for 15 min and scanned with the Odyssey Infrared Imager (LI-COR Biosciences).

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