Runx1

Cell lysates were prepared as described (Hu et al., 2009 (link)). Western blotting was performed as described (Hu et al., 2009 (link)). Antibodies used in the studies including CDK6 from Santa Cruz, RUNX1 from Abcam, Vinculin from Cell Signaling (Cat. 4,650), and α-Tubulin from Sigma.

20 μg of protein extracts in Laemmli buffer were run on a 4–20% gradient pre-cast gel (Bio-Rad) and transferred to nitrocellulose using Turbo transfer packs (Bio-Rad). Membranes were blocked using 5% milk in TBS-T, then RUNX1 (C-terminal: ab23980, 1:3,000; Abcam or N-terminal: sc-8563 N-20, 1:250; Santa Cruz Biotechnology) or anti-HA (H6908, 1:1,000; Sigma-Aldrich) was applied overnight at 4°C in 5% milk in TBS-T. After washing in TBS-T, this was followed with HRP-conjugated anti-rabbit or anti-goat antibody (Cell Signalling Technologies), and enhanced chemiluminescent reagent (Amersham) was applied and the blot was visualised using a GelDoc system (Bio-Rad). For loading controls, the membranes were stripped using Restore Stripping Buffer (Thermo Fisher Scientific) and GAPDH (ab8245; Abcam) was applied and visualised as above.

Cells were lysed in RIPA buffer and boiled in Laemmli sample buffer for 5 min before fractionation by 10% SDS-PAGE. After transfer to nitrocellulose membranes over 1.5 h at 65 V, blots were blocked with 5% milk in PBS containing 0.1% Tween-20 (pH 7.5) and incubated with p45 NF-E2 (1:1000, ref. 39 (link).), FOXP3 (eBioscience, catalog #14-5773), RUNX1 (Abcam, catalog #23980), or FLI1 (Abcam, catalog #15289) Ab overnight at 4 °C. Blots were washed in PBS-Tween, incubated with horseradish peroxidase-conjugated goat anti-rabbit IgG (Santa Cruz, catalog #sc-2054, 1:2,000), and exposed briefly to chemiluminescence reagents (Santa Cruz, catalog #sc-2048).

ChIP-qPCR assays were performed as previously described (Lan et al., 2007 (link)). Briefly, cells were cross-linked with 1% paraformaldehyde for 10 min at room temperature (RT), and immediately quenched by adding 0.125 M glycine. Collect cell pellet and shear the chromatin into 0.2∼1 kb fragment with sonication at 4°C for 25 min (Bioruptor, 90% power, 15 s on, 45 s off). Then, chromatin was immunoprecipitated at 4°C for 3 h with the antibody against Flag (Sigma), RUNX1 (Abcam), or mouse IgG (CST). Antibody-chromatin complexes were pulled-down using protein A beads (GE) for 1 h at 4°C with rotation. Then the beads with specific binding DNA was washed 3 times with high salt buffer (50 mM HEPE NaOH pH 7.5, 500 mM NaCl, 1 mM EDTA, 0.1% Na-Deoxycholate, 1% TritonX100), 2 times with low salt buffer (10 mM TrisCl PH8.0, 250 mM LiCl, 1 mM EDTA, 0.5% NP40, 0.5% Na-Deoxycholate), 1 time with TE buffer. After cross-link reversal and proteinase K (TaKaRa) treatment, DNA fragments were purified with PCR Purification Kit (QIAGEN) and further analyzed by real-time quantitative PCR using the primers as listed in Supplementary Table 2.

Acutely dissected DRGs were lysed in ice-old FA-M2 Lysis Buffer (50 mM Tris HCl, 150 mM NaCl, 1 mM EDTA, 1% Triton X-100, pH 7.5) supplemented with a protease inhibitor cocktail (Sigma;1:100 dilution) by sonication. Clarified lysates were either subjected to Flag immunoprecipitation for co-immunoprecipitation experiments or processed directly for SDS-PAGE. Immunoprecipitation of Flag-CBFβ and its associated proteins was done using anti-Flag M2 affinity gel (Sigma) according to the manufacturer’s instructions. Immunoblotting was performed using antibodies against Runx1 (Abcam, 1:5000), CBFβ (1:1000, Santa Cruz), Histone 3 (1:1000, Cell Signaling Technology) and βIII-Tubulin (1:1000, Covance), as described (Kuruvilla et al., 2000 (link)).