Fig. 1. Effects of oleic acid on the stability of ADPH variants in stably transfected HEK 293 cells. (A) Immunoblot analysis of parental 293 cultures and cultures stably expressing ADPH variants following a 20-hour incubation in the absence (C) or presence of 300 µM oleic acid (OA). Parental 293 culture extracts were immunoblotted with chicken anti-human TIP47, extracts of cultures expressing ADPH were immunoblotted with guinea pig anti-mouse ADPH, and extracts of cultures expressing VSV-tagged forms of ADPH were immunoblotted with mouse anti-VSV. Immunoblots of whole gels are shown to demonstrate specificity and the absence of breakdown products. The boxed regions beneath show the results of immunoblots that were stripped and re-probed for β-actin to control for sample loading. (B) Representative anti-VSV immunoblots of cultures expressing ADPH[fl]-VSV or 2,3 ADPH-VSV following a 20-hour incubation in the indicated concentrations of OA. Anti-β-actin immunoblots are shown beneath the anti-VSV immunoblots. (C) Quantitation of the immunoblots in B. The data show relative steady-state levels of ADPH[fl]-VSV (white bars) and 2,3 ADPH-VSV (black bars) normalized to β-actin. The values are means ± s.d. for triplicate cultures. (D) Effects of OA and/or triacsin C on ADPH[fl]-VSV and 2,3 ADPH-VSV levels. Representative anti-VSV immunoblots of extracts of ADPH[fl]-VSV or 2,3 ADPH-VSV cell lines following a 20-hour incubation in unsupplemented basal culture media (Cont), or in basal media supplemented with 300 µM OA, 5 µM triacsin C (Triac-C), or with 300 µM OA plus 5 µM triacsin C (Triac-C + OA). β-actin loading controls for each sample are shown beneath the anti-VSV blots. Representative immunoblots are from a single well of triplicate cultures treated as indicated. All experiments were repeated at least twice, with similar results.