β mercaptoethanol

Cells were harvested after trypsinization and quenching with medium, washed once with PBS, and lysed with RIPA buffer (Thermo Scientific) supplemented with protease and phosphatase inhibitors (Thermo Scientific). The cell lysates were sonicated using a Branson Sonifier 450 until the viscosity of lysates disappears and centrifuged at 14,000 rpm (17,968 xg) for 5 min. The supernatants were subjected to the DC Protein Assay Kit (BioRad Laboratories). SDS-PAGE samples were prepared by mixing the supernatants and 4x Laemmli buffer/10% beta-mercaptoethanol (BioRad Laboratories) and followed by boiling at 95°C for 5 min. Equivalent amounts of protein were resolved by SDS-PAGE and then transferred to a nitrocellulose membrane using the Trans-Blot Turbo Transfer system (BioRad Laboratories). Membranes were stained with Ponceaus S, blocked with 5% milk/TBS-T (Tris-Buffered Saline/0.1% Tween 20) for 30–60 min, and incubated with primary antibodies, followed by secondary HRP-conjugated antibodies. Blots were developed using a chemiluminescent substrate (SuperSignal West Pico PLUS or SuperSignal West Femto Maximum Sensitivity Substrate; Thermo Scientific) and imaged with an Azure 300 (azure biosystems).

The OCI-AML3 NPM1c-degron 2 cell line was cultured to 10 million for each replicate. Then cells were treated with DMSO or 500 nmol/L dTAG-13 for 24 hours. Cells were extracted with 0.1% NP-40 buffer (50 mmol/L HEPES-NaOH, pH 7.4, 150 mmol/L NaCl, 1 mmol/L EDTA, 2.5 mmol/L EGTA, 10% glycerol, 1× protease inhibitor, 50 mmol/L NaF, 1 mmol/L DTT, 0.1% NP40; Thermo Fisher) for 30 minutes at 4°C with rotation as previously described (60 (link)). The cell extract was centrifuged at 16,600 × g for 15 minutes at 4°C. The supernatant was collected with 50 μL as input. Bio-Isoxazole (Sigma) was added to the remaining supernatant to a final concentration of 33 μmol/L. The remaining protein extract with Bio-Isoxazole was then incubated at 4°C with slow rotation for 1 hour. The precipitated protein was collected by centrifugation at 14,000 × g for 5 minutes, and 50 μL of the supernatant was then taken out as supernatant. Precipitated protein was then washed with 0.1% NP-40 extraction buffer 2 times with centrifugation at 14,000 × g for 5 minutes. Then 2× laemmli buffer with 5% beta-mercaptoethanol (Bio-Rad) was used to dissolve the protein precipitation with heating at 95°C for 5 minutes. An equal volume of 2× laemmli buffer was added to the input and supernatant samples and boiled at 95°C for 5 minutes. Boiled protein samples were then used in PAGE gel separation and immunoblot analysis.