Glutathione acceptor beads

Hypoxanthine, sorbitol, percoll, MG132, Epoxomicin, Lactacystin, Clasto-Lactacystin β-lactone, Gliotoxin, MG115, EDTA, sodium phosphate dibasic, monosodium phosphate, Tris HCl, sodium chloride, Tween-20, sodium pyrophosphate, glycerol-2-phosphate, saponin, bovine serum albumin (BSA), dithiothreitol (DTT) and phenylmethylsulphonyl fluoride (PMSF) were purchased from Sigma. Glutathione acceptor beads, protein A donor beads, DELFIA enhancement solution and DELFIA secondary antibody (Eu-N1 rabbit Anti-mouse-IgG) came from Perkin Elmer. NP-40 was purchased from Calbiochem, sodium fluoride from Panreac, antibody anti-ubiquitin P4D1 from Santacruz, deubiquitylases inhibitor PR-619 came from Merck, complete mini EDTA protease inhibitor cocktail from Roche, antibody anti-ubiquitin FK2 from Enzo, biotin-TUBEs from Life sensor, RPMI 1640 medium from Gibco, AlbuMAX II from Invitrogen, bortezomib from Selleckchem, enhanced chemiluminescence (ECL) from GE Healthcare and PBS from Oxoid. Atovaquone was prepared in house.

The purified claudin-4 samples were diluted to 40 nM (final 10 nM) in the reaction buffer [50 mM Tris-HCl buffer (pH 7.0), containing 0.05% βDDM, 0.002% CHS, and 400 mM NaCl], and 5 μl aliquots were dispensed per well in a 384-well plate (AlphaPlate-384, PerkinElmer) on ice. In the blank well, an equal volume of the reaction buffer was dispensed. The purified GST-tagged C-CPE was also diluted to 160 nM (final 40 nM) in the reaction buffer, and a 5 μl aliquot of diluted GST-tagged C-CPE was added to all wells, mixed by shaking 30 sec, and then incubated on ice. Two hours later, a 5 μl aliquot of 80 μg/ml (final 20 μg/ml) Glutathione acceptor beads (PerkinElmer) in the reaction buffer was added to all wells, mixed by shaking 30 sec, and then incubated on ice for 1 hr in the dark. Finally, a 5 μl aliquot of 80 μg/ml (final 20 μg/ml) Anti-FLAG donor beads (PerkinElmer) in the reaction buffer was added to all wells, mixed by shaking 30 sec, and then incubated on ice for 1 hr in the dark. Before measurement, the plate was incubated at room temperature for 20 min in the dark. Alpha counts were measured by EnVision (PerkinElmer), using the standard AlphaScreen settings.

The binding assays were performed using an Hsp90 (C-terminal) Inhibitor Screening Kits (cat. 50314 or 50317) purchased from BPS Bioscience. The assay was performed according to the manufacturer's protocol and utilised AlphaLISA® technology (PerkinElmer). The test compounds were dissolved in 100% DMSO and diluted with water to the desired concentration, so that the final dilution was dissolved in 5% DMSO with water. Two microlitres of the dilution was added to a 10 µL reaction so that the final concentration of DMSO was 1% in all reactions. The reactions were conducted at room temperature for 30 min in a 10 µL mixture containing assay buffer, 6 ng (24 nM) of a C-terminal Hsp90β (UniProt P08238, a.a. 527–724) or C-terminal Hsp90α (UniProt P07900, a.a. 535–732), 40 ng (60 nM) Cyp40, and the test compound. Note: for the full-length Hsp90 binding assays, C-terminal Hsp90 was replaced with 24 nM of full-length Hsp90α (Abcam cat # ab48801). After the 30 min incubation, 10 µL of buffer containing 20 µg/mL glutathione acceptor beads (Perkin Elmer) were added to the reaction mix and incubated for 30 min in the dark. Ten microlitres of 40 µg/mL streptavidin donor beads (Perkin Elmer) were then added and the final 30 µL mixture was incubated for 1 h in the dark. The AlphaLISA^® signal was measured using a Tecan F200 Pro multimode plate reader.