HEK293 cells were transfected in combination of Halo-SMAD4, HA-tagged ubiquitin, and/or SNAP-Flag-BRK-YF, and the cells were treated with 10 μM MG132 for 6 hours. Denatured cell extracts were prepared using denaturing buffer containing 1% SDS, 50 mM tris-HCl (pH 7.5), 150 mM NaCl, 1% Triton X-100, 0.1% sodium deoxycholate, 0.1 mM benzamidine HCl, 55 μM phenanthroline, 10 μM bestatin, 20 μM leupeptin, 5 μM pepstatin A, and 1 mM PMSF. Then, the cell lysates were and boiled for 10 min before incubation with primary mouse anti-SMAD4 antibody, followed by Dynabeads Protein G (Invitrogen) magnetic beads conjugation and immunoblotting with anti-HA antibody to detect ubiquitinated SMAD4.

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