Imagej software 4

Forebrain samples were homogenized in RIPA lysis buffer (Santa Cruz Biotechnology) and supernatants from the homogenates were collected after centrifugation at 20817 g, at 4°C for 30 minutes. The protein concentration was determined using a detergent compatibility assay (Bio‐Rad). Thirty micrograms of protein per sample were loaded, run for 30 minutes at 80 V, then 60 minutes at 120 V, followed by protein transfer onto nitrocellulose membranes at 0.45 mm for 120 minutes (Bio‐Rad). Membranes were incubated for 2 hours in 5% nonfat milk in Tris‐buffered saline containing 0.1% Tween 20. The following primary antibodies were incubated overnight at 4°C: anti‐IRP1 and anti‐IRP2 (1:1000; Abcam), anti‐NCBE (1:500; Origene) and anti–β‐Actin (1:1000; Santa Cruz Biotechnology). The membranes were washed and then incubated with the appropriate secondary antibodies (1:4000, Santa Cruz Biotechnology) for 1 hour at room temperature. Enhanced chemiluminescent solution (GE Healthcare and Life Science) was then applied to the membranes and the protein bands were exposed onto radiography films. ImageJ software (4.0, Media Cybernetics) was used to analyze the relative density of the resultant protein immunoblots.