Immunoblot analysis.

After growth in the indicated medium, cells corresponding to 0.5 OD₆₀₀ were collected by centrifugation; resuspended in 1× phosphate-buffered saline (PBS) (KD Medical), 7 μl of 2× Laemmli buffer (Bio-Rad), and 2 μl of β-mercaptoethanol; and 10 μl was loaded on a Mini-Protean TGX 5% to 20% Tris-glycine gel (Bio-Rad) and run in 1× Tris glycine-SDS (KD Medical) buffer. The proteins were electrotransferred to nitrocellulose membranes (Invitrogen) for 1 h at 100 V. Membranes were blocked with 5% nonfat milk (Bio-Rad) in 1× PBS with 0.1% of Tween 20 (PBS-T) for 1 h and probed with a 1:1,000 dilution of anti-HA antiserum (Abcam) in the same PBS-T buffer with 5% milk for 1 h. After the incubation with the anti-HA antiserum, membranes were incubated with a 1:20,000 dilution of horseradish peroxidase (HRP)-labeled goat anti-rabbit antibody (Pierce). All blots were washed 4× with PBS-T and then developed with an Amersham ECL Western blotting detection kit (GE Healthcare).