Protein a agarose

For protein–protein interaction analysis, HEK293T cells (4 × 10⁶) were seeded into 10‐cm dish and then transfected with various plasmids. After 48 h, cells were harvested and lysed in immunoprecipitation buffer (50 mm Tris, pH 7.4; 150 mm NaCl; 2 mm EDTA; 3% glycerol; 1% NP‐40; complete, EDTA‐free protease inhibitor cocktail tablets). After sonication, cell lysates were centrifuged at 13 000 g for 10 min at 4 °C. Thereafter, supernatants were incubated with indicated antibodies (1 μg) for 3 h at 4 °C, and then rotated with Protein A‐agarose (Merck Millipore, Darmstadt, Germany) for 3 h at 4 °C. After being washed with lysis buffer for 6 times, the immunoprecipitation materials were boiled in 40 μL 2 × SDS loading buffer and subjected to western blotting using indicated antibodies.

Eight-day-old transgenic seedlings of 35S::SPL9-HA or 35S::SPL15-HA were cross-linked with 1% formaldehyde and ground in liquid nitrogen. The chromatin complex was prepared following the method of Xie et al.^{24 (link)}. In brief, the supernatant was pre-cleared with 40 μl Protein-A-Agarose (16-157, EMD Millipore Corp.) and incubated at 4 °C for 1 h. Then the supernatant was moved into a microtube and 5 μl HA tag monoclonal antibodies (CB100005M, Cali-Bio) were added. After incubation at 4 °C for overnight with gentle agitation, 40 μl Protein-A-Agarose was added and incubated for 2 h. After washing, the immuno complex was eluted from the agarose beads. The precipitated DNA was then recovered and quantified using quantitative PCR with their individual primer pairs. The values were standardized to the input DNA to obtain the enrichment fold. PP2A was used as the internal control.

The enzymatic activity of cell surface-expressed TPO in the studied cell lines was analyzed as we had previously described [31 (link)]. Briefly, cells were grown 48 h in a medium supplemented with 20 mM hemin (Sigma-Aldrich). After washing with PBS, cells were incubated with reaction mixture containing Amplex Ultra Red (Life Technologies) in the presence of superoxide dismutase (SOD), KI, and H₂O₂ (Sigma-Aldrich). Reaction was stopped at 1-minute intervals by addition of catalase and SOD. The fluorescence was measured in the Synergy H4 hybrid multi-mode microplate reader (BioTek, USA) [31 (link)]. The enzymatic activity of TPO expressed in tissues was determined using a luminol-based assay described by Jomaa and collaborators [25 (link), 32 (link)], with some modifications. Briefly, breast tissue lysates (200 μg) were immunoprecipitated with mAb A4 (6 μg) and protein A agarose (Merck Millipore, Darmstadt, Germany). 50 μl of resin-bound TPO in 0.1 M Tris-Cl (pH 8.6) was incubated with 150 μl of 1.3 M glycine-NaOH (pH 9.0), 1.3 mM EDTA in a 96-well plate for 5 minutes. The reaction was initiated by adding 20 μl of 400 μM luminol (Sigma-Aldrich) in 1 M glycine-NaOH (pH 9.0), 1 mM EDTA, followed by the supplementation with 5 μl of 80 mM H₂O₂. Luminescence intensities were measured in the Synergy 2 instrument (BioTek).

The lysates of BM cells (2 × 10⁶) from 8-week-old mice were prepared for the ChIP assay^{70 (link)}. BM cell lysates were sonicated and immunoprecipitated with the anti-Bmi1 (Santa Cruz Biotechnology, Inc. sc-10745, 2 μg sample⁻¹), anti-Cbx7 (Santa Cruz Biotechnology, Inc. sc-70232, 2 μg sample⁻¹), anti-H2AK119ub (Cell Signaling Technology 8240, 2 μg sample⁻¹), anti-H3K9ac (Merck 06-942, 2 μg sample⁻¹), anti-H3K4me3 (Abcam ab12209, 2 μg sample⁻¹), anti-H3K27me3 (Merck 07-449, 2 μg sample⁻¹), anti-Phc2 (Santa Cruz Biotechnology, Inc. sc-160664, 2 μg sample⁻¹), and anti-Ring1b (Cell Signaling Technology 5694, 2 μg sample⁻¹) Abs. Additionally, GSK126-treated OP9 cell lysates were sonicated and immunoprecipitated with the anti-Bmi1, anti-Phc2, and anti-Ring1b Abs. After immunoprecipitation, immune complexes were collected with protein A agarose (Merck GE17-0963-03) and extracted with an extraction buffer (1% SDS, 0.1 M NaHCO₂). DNA cross-links were reversed by heating to 65 °C for 8 h. DNA was extracted with phenol/chloroform and precipitated with ethanol. DNA isolated from an aliquot of total nuclear extract was used as the loading control for PCR (input control). qPCR was performed as described above. Data are presented after normalizing each immunoprecipitated DNA Ct value to 10% of the input DNA Ct value.

The cell lysate was prepared using radioimmunoprecipitation assay buffer containing protease inhibitor (Roche, Basel, Switzerland) and then immunoprecipitated with 2 μg anti-HDAC6 (#7612, Cell Signaling Technology, Inc.) or IgG (sc-2025/2027, Santa Cruz Biotechnology, Inc.) at 4 °C overnight, followed by incubation with protein A agarose (Merck Millipore, Bedford, MA, USA) at 4 °C for 1 h. For the immunoprecipitation of FLAG-tagged CNOT6, anti-FLAG M2 affinity gel (Sigma-Aldrich Corp.) was used following the manufacturer’s instructions. The immune complexes were detected by Western blot analyses with anti-CNOT6 (sc-81,231, 1:1000, Santa Cruz Biotechnology, Inc.), anti-HDAC6 (#7612, 1:1000, Cell Signaling Technology, Inc.), and anti-acetyl-lysine (GTX80693, 1:1000, GeneTex Inc., Irvine, CA, USA) antibodies.