Na943

Cell lysates were prepared in a buffer containing 20 mM Tris (pH 7.4), 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1% Triton X-100, 25 mM sodium pyrophosphate, 1 mM NaF, 1 mMβ-glycerophosphate, 0.1 mM sodium orthovanadate, 1 mM PMSF, 2 μg ml⁻¹ leupeptin and 10 μg ml⁻¹ aprotinin (Sigma Aldrich). A total of 50 μg of cell lysates were separated on SDS–PAGE gel and transferred onto Amersham hybond ECL nitrocellulose membranes (Amersham, Baie d'Urfe, QC, Canada). The membranes were blocked with 5% skimmed milk and then incubated with the indicated antibodies at 4 °C overnight. Appropriate HRP-conjugated secondary antibodies were incubated for 1 h at room temperature. Signals were detected using an ECL Western Blotting Kit (Amersham). The primary and secondary antibodies and the concentrations used were as follows: mouse anti-ALDH1A1 (44/ALDH, 1 : 2000, BD Biosciences, Mississauga, ON, Canada), rabbit anti-ALDH3A1 (B-8, 1 : 500, Santa Cruz Biotechnology, Santa Cruz, CA, USA), goat anti-actin (C-11, 1 : 1000, Santa Cruz Biotechnology), donkey anti-goat (SC2033, 1 : 3000, Santa Cruz Biotechnology), sheep anti-mouse (NA931, 1 : 3000, GE Healthcare, Mississauga, ON, Canada) and donkey anti-rabbit (NA943, 1 : 3000, GE Healthcare).