Figure 2.

HSC70/HSP70 inactivation blocks CTA1 translocation to the cytosol. A, CHO cells were incubated with varying concentrations of CT for 2 h in the absence (circles) or presence (squares) of 20 μm PES before intracellular cAMP levels were determined. Data are presented as mean ± S.E. from 4 independent experiments with triplicate samples. B, CHO cells were mock transfected or transfected with the ssCTA1/pcDNA3.1 expression plasmid that directs CTA1 for co-translational insertion into the ER. After a 1-h incubation with [³⁵S]methionine, the ER-to-cytosol export of CTA1 was monitored through immunoprecipitation of organelle pellets (P) and cytosolic supernatant (S) fractions generated from untreated (−PES) or PES-treated cells. The percentages of radiolabeled CTA1 detected in the cytosolic fractions are presented as box-and-whisker plots (n = 4). The asterisk indicates a statistically significant difference from the control (p = 0.02, Student's t test).