Figure 10.
Interaction between CERT and VAP is sterol and OSBP sensitive. (A) shNT and shOSBP cells received NA or were treated with 6 μM 25OH (for 2 h), 5 mM MβCD (for 30 min), or the control solvents ethanol (EtOH) and water. Cells were incubated with 1 mM dithiobis succinimidyl propionate, extracted, and immunoprecipitated (IP) with rabbit polyclonal antibodies against OSBP, VAP, or a nonimmune rabbit serum (NI). Immune complexes were recovered on protein A-Sepharose, resolved by SDS-10% PAGE, and immunoblotted (IB) for CERT. The input lane contains 25% of the cross-linked cell extract used for immunoprecipitation. (B) Blots from three independent experiments were scanned by densitometry, and VAP association with CERT was quantified (relative to solvent-treated controls) in shNT (light gray bars) or shOSBP cells (dark gray bars).