Bio isoxazole

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.