Aldh1a2

Cells were washed with PBS and solubilized in PRO-PREPTM protein extraction solution (iNtRON biotechnology, Korea) for 15 min at 4°C. Cell debris was eliminated by centrifugation at 14,000 rpm for 10 min at 4°C. The supernatant was analyzed for protein content by the Bradford method, and 20 μg of protein was separated on a 10% polyacrylamide gel. The protein was transferred to a hydrobond ECL nitrocellulose membrane (Amersham Biosciences; Piscataway, NJ) and incubated overnight at 4°C with polyclonal antibody against ALDH1A1 (Abcam; Cambridge, MA; 1:1000 dilution), ALDH1A2 (Abcam; Cambridge, MA; 1:1000 dilution), ALDH1A3 (Genetex; Cambridge, MA; 1:1000 dilution), or ALDH3A1 (Cusabio; CA; 1:1000 dilution) in Tris-buffered saline (50 mM Tris, pH 7.5, 150 mM NaCl) with 0.05% polysorbate-20 containing 5% skim milk. After washing three times for 10 min with Tris-buffered saline with Tween 20, the membrane was incubated with secondary antibodies for 1 h at room temperature. Immune reactive protein was detected with an enhanced chemiluminescence kit and quantified using Quantity One software (Bio-Rad Laboratories, CA).