Figure 4

Effect of the pharmacological inhibition of Hsp90, Cyps and FKBPs on the uptake of DTA into the cytosol of HeLa cells treated with nicked biotin-labeled DT. (a) Detection of biotin-labeled DTA in the cytosol of HeLa cells. 10⁶ cells were suspended in 100 µl of serum-free medium and incubated for 1 h at 37 °C with 1 µg of nicked biotin-labeled DT in the presence or absence of Baf A1. For control, cells were left untreated. Subsequently, the cells were pelleted, washed and the cytosolic fractions were extracted by digitonin-treatment for subsequent pull-down of biotinylated proteins at 4 °C with streptavidin-agarose beads. The precipitated biotin-proteins were removed from the agarose beads by heating in SDS-sample buffer, separated by SDS-PAGE and detected by Western blotting with streptavidin-peroxidase (upper panel). To confirm that comparable protein amounts were used for the precipitation step, equal aliquots of the cytosolic fractions were removed prior to precipitation, subjected to SDS-PAGE, blotted onto nitrocellulose and stained with Ponceau S (lower panel). The Western blot panel was cropped for presentation purposes only. (b) Less DTA reaches the cytosol of DT-treated HeLa cells after pre-treatment of cells with Rad, CsA or FK506. HeLa cells were pre-incubated for 30 min at 37 °C with either Rad (20 µM), CsA (20 µM) or FK506 (10 µM) and then treated in suspension with nicked biotin-labeled DT. Biotinylated DTA was precipitated from digitonin-extracted cytosolic fractions and detected by Western blotting exactly as described in a. The upper panel shows the Western blot analysis of DTA (left lane, nicked B-DT as running control). Lower panel: Confirmation of comparable input of cytosolic proteins into the precipitation step by Western blotting against Hsp90. The Western blot panel was cropped for presentation purposes only.