Cell Culture and Western Blot
CHO cells were used to heterologously express dSlo and Slob. In brief, CHO cells were maintained in Ham's F-12 nutrient mixture (Invitrogen) containing 10% FBS (Invitrogen) and 100 U/ml penicillin and streptomycin (Invitrogen). Plasmids were transfected into CHO cells using Lipofectamine 2000 reagent (Invitrogen). 1 d after transfection, cells were harvested, washed, and lysed in lysis buffer (25 mM Tris-HCl, pH 7.5, 150 mM NaCl, 50 mM NaF, 50 mM KCl, 1% CHAPS, 1 mM phenylmethylsulfonyl fluoride, 2 mM DTT, 1 mg/ml each aprotinin, leupeptin, and pepstatin A). Supernatants of the cell lysates, separated by centrifugation at 16,000 g for 10 min, were used for analysis of protein expression, or were incubated for 2 h with anti-Slob antibody with rotation at 4°C for immunoprecipitation. After this incubation, protein A/G agarose beads were added, and the incubation was allowed to proceed with rotation at 4°C overnight to precipitate the immunocomplex. Beads were then isolated by centrifugation at 5,000 g for 10 min. After four washes with PBS, the beads were used for Western blot analysis.
Proteins in the cell lysates or on the beads were solubilized in SDS loading buffer as described previously (Zeng et al., 2004) and loaded onto 4–15% gradient or 7.5% SDS polyacrylamide gels (Bio-Rad Laboratories), separated by gel electrophoresis, and transferred to nitrocellulose membranes for Western blotting. After blocking in TBST (Tris-HCl–buffered saline with 0.1% Tween 20) containing 5% nonfat milk, membranes were incubated with primary antibody (Schopperle et al., 1998) for 1 h at room temperature. After washes with TBST, membranes were incubated with horseradish peroxidase–conjugated donkey anti-rabbit antibody at room temperature for 1 h. Labeled proteins were visualized using an enhanced chemiluminescence reagent according to the manufacturer's specifications (GE Healthcare), and the images were acquired with a Kodak Image Station 4000R and Kodak Imaging Software (version 4.0).