E 3652

Cells were pretreated for 8 h with epoxomicin (100 nM, Sigma-Aldrich E 3652) and then lysed by 600 µL of buffer containing 6 M guanidine hydrochloride, 0.1 M NaH₂PO₄/Na₂HPO₄ pH 8.0 (ratios calculated by http://clymer.altervista.org/buffers/phos2.html) 10 mM imidazole. Lysate was collected, sonicated, and heated to 95 °C for 6 min. Lysates were then centrifuged 16,200 g/20 min, and 500 μL of cleared lysate was used for incubation with magnetic His-beads (5 μL slurry) overnight (28-9799-17, GE-Healthcare). Pulldown samples were washed by buffer containing 8 M UREA, 0.1 M NaH₂PO₄/Na₂HPO₄ pH 6.3, 0.01 M Tris pH 8.0, 20 mM Imidazole, by using magnetic stand. After the last wash supernatant was completely removed and 100 μL of 1× sample buffer was added to the beads. Pulldown samples were heated to 95 °C for 6 min prior to loading. Lysates for input assessment were prepared by removal of SDS-PAGE incompatible guanidine-hydrochloride, by precipitation of the lysate (approximately 100 uL) by 900 μL of 100% ethanol overnight at −20 °C. Precipitated protein was then centrifuged at 16200 RCF for 20 min, supernatant was discarded, 500 μL of 90 % of ice-cold ethanol was added, vortexed, incubated at −20 °C for 20 min, centrifuged 16,200 RCF for 20 min, the supernatant was again discarded and the pellet was dried out and resuspended in 100 μL of 1× sample buffer.

Cells were transfected with the plasmid encoding polyhistidine-tagged ubiquitin, RNF43-HA, or enzymatically inactive RNF43, protein of interest, and cultured overnight. Next, cells were treated with 0.2 µM epoxomicin (E3652, Sigma) for 4 hr and lysed in the buffer containing 6 M guanidine hydrochloride (G3272, Sigma), 0.1 M Na_xH_xPO₄ pH 8.0, and 10 mM imidazole (I5513, Sigma), sonicated, and boiled. Insoluble fraction was removed by the centrifugation (16,000 g, room temperature [RT], 10 min). For the pull down of tagged proteins, 10 µl of equilibrated in lysis buffer His Mag Sepharose beads Ni (GE28-9799-17, GE Healthcare) was added to each sample and kept on a roller overnight. Then, the beads were washed three times in the buffer containing 8 M urea (U5378, Sigma), 0.1 M Na_xH_xPO₄ pH 6.3, 0.01 M Tris, and 15 mM imidazole, resuspended in 100 μl of western blot sample buffer, boiled for 5 min, and loaded onto SDS-PAGE gel. Approximately 10% of cellular lysate was used as a transfection control after ethanol precipitation and resuspension in the western blot sample buffer.