Sample buffer solution with reducing reagent 6 for sds page

Western blotting was performed to confirm the expression of purified GST-tagged p11.5×1, p11.5×2, and p11.5×3 recombinant proteins by transformed bacteria. The bacterial lysates containing recombinant proteins were heated at 100 °C for 5 min in SDS sample buffer (Sample Buffer Solution with Reducing Reagent (6×) for SDS-PAGE; Nacalai Tesque), subjected to electrophoresis in denaturing gels (Mini-PROTEAN TGX Gels 4%–20%; Bio-Rad Laboratories, Inc., Hercules, CA, USA), and transferred to PVDF membranes. The membranes were blocked with Bullet Blocking One for Western Blotting (Nacalai Tesque) for 20 min and reacted with the indicated antibodies for at least 1 h at room temperature or overnight at 4 °C. These membranes were then reacted with secondary antibody conjugated with peroxidase (Jackson) and visualized using Chemi-Lumi One L or Chemi-Lumi One Super (Nacalai Tesque) with the chemiluminescence imaging system (ChemiDoc Touch; Bio-Rad Laboratories).

To assess endogenous ubiquitination of PLZF and SALL4, 2 × 10⁷ lt-NES cells were pretreated with DMSO or 1 μM MLN4924, treated with DMSO or 10 μM pomalidomide for 7 h, and then lysed with Pierce IP Lysis Buffer (ThermoFisher Scientific) containing 10 mM N-ethylmaleimide (ThermoFisher Scientific), 10 μM PR-619 (Nacalai Tesque), 10 μM MG132 (Peptide Institute), and protease/phosphatase inhibitor cocktail. Ubiquitinated proteins were pulled down by incubating lysates with Ubiquilin 1 tandem UBA (TUBE2) agarose (Boston Biochem) for 2 h at 4 °C and washing the beads 3 times with IP lysis buffer. Bound proteins were eluted by incubating the beads with Sample Buffer Solution with Reducing Reagent (6×) for SDS-PAGE (Nacalai Tesque) at 95 °C for 5 min and separated by SDS-PAGE, followed by immunoblotting using antibodies against PLZF, SALL4, and Ubiquitin.