Anti pu 1 t 21

After lysis with RIPA buffer containing 0.15 M NaCl, the lysate was centrifuged. The pellet was resuspended in RIPA buffer containing 0.4 M NaCl. The supernatant was then used for EMSA analysis. Four micrograms of the nuclear extracts were incubated with ³²P-labeled probe for 20 min in binding buffer (50 mM Tris-Cl [pH 7.5], 20% glycerol, 5 mM MgCl₂, 2.5 mM EDTA, 2.5 mM DTT, 250 mM NaCl, and 0.25 mg/ml poly[dI-dC]·poly[dI-dC]). For supershift analysis, extracts were incubated with anti-RUNX1 (N-20, Santa Cruz Biotechnology), anti-PU.1 (T-21, Santa Cruz Biotechnology), or control antibodies (Sigma-Aldrich), followed by a radiolabeled probe. Reaction mixtures were analyzed through separation on 6% polyacrylamide gels. RUNX1 and PU.1 site probes are as follows: RUNX1 upstream site, 5'-TTTCAGGAGTGGTGACGCCT-3'; RUNX1 downstream site, 5'-AGCAGGTACCACTGGTCTTC; PU.1 site, 5'-GGAGGGAGGAGAGGAAGGTA-3.'

Cells were lysed in RIPA buffer (50 mM Tris-Cl [pH 7.4], 0.1% NaN₃, 1% Nonidet P-40, 0.25% sodium deoxycholate, 1 mM EDTA, 1 mM Na₃VO₄, 1 mM NaF, and protease inhibitor mixture) supplemented with 0.4 M NaCl. Lysates were centrifuged, and the resulting supernatants were subjected to Western blot analysis. Thirty micrograms of the cell lysates were resolved with SDS-PAGE and then transferred to polyvinylidene difluoride membranes. After the membranes were blocked with 5% nonfat dry milk, they were probed with anti-RUNX1 (N-20, Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-PU.1 (T-21, Santa Cruz Biotechnology), and anti-GAPDH Ab (Santa Cruz Biotechnology). The membranes were incubated with an anti-goat HRP-conjugated Ab (Santa Cruz Biotechnology) for RUNX1 and an anti-rabbit HRP-conjugated Ab (Cell Signaling Technology, Beverly, MA, USA) for PU.1. Immunostained proteins were visualized using an ECL detection system (Amersham Biosciences, Buckinghamshire, UK).