U5378

Fifty micrograms of protein were digested in 8 M urea buffer (8 M urea (U5378, Sigma‐Aldrich), 100 mM Tris 8.5 (15,504,020, Thermo Scientific), 10 mM dithiothreitol (D9779, Sigma‐Aldrich)) using the filter‐aided sample preparation (FASP) protocol. Briefly, proteins were added to a 30 kDa cut‐off filter (MRCF0R030, Millipore) and centrifuged at 11,000 rotations/min at 20°C for 15 min. Iodoacetamide (I1149, Sigma‐Aldrich) (50 mM) in urea buffer was used to alkylate the proteins at 20°C for 15 min. After a few washes with urea buffer and 50 mM ammonium bicarbonate (ABC; 09830, Sigma‐Aldrich) buffer, 100 ng of trypsin (V5280, Promega) in 50 mM ABC buffer was used to digest proteins in a wet chamber overnight at 37°C. Peptides were extracted using 50 mM ABC buffer and acidified with trifluoroacetic acid (TFA; 1,082,620,100, Millipore). After performing the FASP protocol described above, the peptides were separated into 5 fractions (flow through, pH 11, pH 8, pH 5, pH 2). Each sample was measured by LC–MS/MS using a 4 h gradient.

During the experiment, a medium [26 (link)] of the following composition was used: 30 mL of sodium chloride (S9888; Sigma-Aldrich, Munich, Germany), 175.3 g L⁻¹ solution; 68.3 mL of sodium hydrogen carbonate (131638; PanReac Applichem, Barcelona, Spain), 84.7 g L⁻¹ solution; 4.2 mL of potassium chloride (191494; PanReac Applichem, Barcelona, Spain), 89.6 g L⁻¹ solution; 200 mL of hydrochloric acid (170266; LenReaktiv, Moscow, Russia), 37% w/v solution; 10 mL of urea (U5378; Sigma-Aldrich, Munich, Germany), 25 g L⁻¹ solution; 10 mL of calcium chloride dihydrate (121214; PanReac Applichem, Barcelona, Spain), 22.2 g L⁻¹ solution; albumin (CAS Number 9048-46-8; “Sonac Loenen BV,” Netherlands), 1.8 g L⁻¹; and pork bile (48305; Sigma, St. Louis, MO, USA), 6.0 g L⁻¹, pH 8.0.

Fifteen thousand A549 cells were seeded in each well of a 6-well plate and the cells were grown in 80 ppm DDW medium or treated with, 0.5 μm CAMP, 2 nm PCTL or 0.5 μm MTX in four replicates. After 48 h treatment, the cells were collected and lysed using 50 mm Tris (741883, Sigma) buffer and 8 m urea (U5378, Sigma), 1% SDS, and protease inhibitor (5892791001, Sigma) at pH 8.5. For the time course experiment, the cells were grown in either NW or 80 ppm DDW and treated with either MTX or PCTL for 4, 15, 26, 38, and 48 h. After protein reduction using 8 mm DTT (10708984001, Sigma) and alkylation using 25 mm IAA (I1149, Sigma), the proteins were precipitated using cold acetone at −20 °C overnight followed by centrifugation and resuspension. Proteins were then digested by Lys C (125–05061, Wako Chemicals GmbH, Neuss, Germany) (1:75 enzyme to protein ratio) at 30 °C for 6 h and trypsin (V5111, Promega) (1:50 enzyme to protein ratio) at 37 °C overnight. After labeling using the TMT-10 reagent (90110, Thermo Fisher Scientific), desalting on C18 Sep-pak columns (WAT054960, Waters, Milford, MA) and fractionation using high pH reversed-phase peptide fractionation kit (84868, Thermo Fisher Scientific) according to manufacturer's instructions, the obtained 10 fractions of peptides in each sample were analyzed by shotgun proteomics using nanoLC-MS/MS.

Fifteen thousand A549 cells were seeded in each well of a 6-well plate and the cells were grown in 80 ppm DDW or NW, and treated with either a vehicle or auranofin at 3 μm in 3 replicates. After 48 h, the cells were collected and lysed in lysing buffer at pH 8.0: 50 mm HEPES (U5378, Sigma) with addition of 8 m urea, 1 mm EDTA (E9884, Sigma), 1% SDS and protease inhibitor. The samples were incubated with 4.4 mmol/L of iodoTMT-126, iodoTMT-127 and iodoTMT-128 (90102, Thermo Fischer Scientific) over night at 37 °C. Free SH and SSH groups were blocked in this stage. After precipitation using methanol/chloroform, the samples were dissolved in 50 mm HEPES buffer including 8 m urea. 10 mm TCEP (T2556, Thermo Fischer Scientific) was used to reduce disulfides at 50 °C for 1 h. After precipitation followed by resuspension, the samples were labeled by iodoTMT-129, iodoTMT-130 and iodoTMT-131 reagents overnight at 37 °C. Afterward, the labeled samples were precipitated and then resuspended for digestion using Lys C (1:75, enzyme to protein ratio) at 30 °C for 6 h and trypsin (1:50, enzyme to protein ratio) at 37 °C overnight. After desalting, the iodoTMT-labeled peptides were enriched using immobilized anti-TMT resin (90076, Thermo Fischer Scientific) according to manufacturer's instructions. The enriched peptides were analyzed by shotgun proteomics using nanoLC-MS/MS.

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.

Formula of melanin bleaching solution was modified based on previous literature.^{48 (link)} Bleaching solution A: 1% H₂O₂ (v/v) (Sigma-Aldrich, H1009), 15% urea (w/v) (Sigma-Aldrich, U5378) in 0.05 M Tris (Sigma-Aldrich, T6066), pH 10.0. Bleaching solution B: 3% H₂O₂ (v/v), 15% urea (w/v) in 0.05 M Tris, pH 10.0.