Figure 2. Intracytoplasmic soluble expression of intrabody-PEST and intrabody-scrambled PEST constructs is increased significantly with respect to intrabody alone when treated with epoxomicin. (A) D5E scFv, designated E; (B) 10H scFv, designated H; (C) D10 scFv, designated D; (D) VH14, designated V. For all intrabodies, the level of soluble protein is significantly higher in epoxomicin treated cells compared with that of vehicle (DMSO) treated cells in Figure 1; Ratio of Figure 2 (+inhibitor) to Figure 1 (no inhibitor) values are shown at the bottom of each graph in Figure 2. Multiple fold increase is not apparent by just looking at the y axis scales of the Figure 1 and 2 because multiple exposures were taken for each treatment. Actin was used as a control to evaluate protein levels across various exposures. ST14A cells were transiently transfected with intrabody, intrabody-PEST or intrabody-scrambled-PEST constructs. Twelve hours prior to harvest cells were treated with 9 μM epoxomicin and 48 h after the transfection cells were harvested, cell lysates prepared, soluble protein separated and run on western blots. Proteins were identified using anti-HA, with actin as a loading control. Proteins were quantified densitometrically and normalized to actin. Inhibitor and non-inhibitor lanes actin bands were used separately from different exposures to calculate ratios, using only non-saturated exposures. At least 3 independent experiments were performed and representative gels are illustrated. One-way ANOVA with Minitab statistical software was used to perform statistical significance. (*p < 0.05, **p < 0.01, compared with intrabody-HA)