Alternatively, a commercially available p-actin monoclonal antiserum (Sigma) was added to the membrane at a dilution of 1:8000 and incubated overnight. The membranes were washed at least four times for 15 min in TBST. The second antibody was then added to the membrane and incubated for 1 h (for H+-ATPase, we used a rabbit anti-chicken immunoglobulin G conjugated to horseradish peroxidase [Sigma]; for p-ac-tin, we used a goat anti-mouse immunoglobulin G conjugated to peroxidase) before being thoroughly washed in TBST.
The H+-ATPase protein was detected using enhanced chemiluminescence (Amersham, Buckinghamshire, U.K.) according to the manufacturer’s instructions. The membranes were then exposed to Hyperfilm-LS (Kodak) until optimal development of the signal was detected. The film was scanned (Snapscan-e-40; AGFA, Middlesex, U.K.) using Scanwise software (AGFA). The image was then mounted in Photoshop 5.5 (Adobe, San Jose, CA).
Data are expressed as means ± SEM. One-way ANOVA was performed followed by the Tukey test. Statistical significance was conducted at a level of confidence of P < 0.05.