ECA1p was mainly localized on the ER of Arabidopsis plants. Membrane (2–6 μg protein) fractionated by a sucrose gradient was separated by SDS/PAGE, blotted, and probed with either polyclonal antibodies against ECA1p (1:500 dilution), anti-CTF2 (37) against the C-terminal end of PM H+-ATPase, AHA2 (1:10,000), or monoclonal antibodies 11A1 against oat calnexin (1:100) and 2E7 (24) against the B subunit of oat V-ATPase (1:500). One experiment is representative of two experiments.