Anti hoxb13

Antibodies of this study were as follows: anti-HBXIP (Abcam, UK), anti-HOXB13 (Abcam), anti-β-actin (Sigma-Aldrich, USA), anti-Flag-tag (Sigma-Aldrich), anti-STAT3 (ImmunoWay Biotechnology Company, USA), anti-GFP (Sigma-Aldrich), anti-GCN5 (Proteintech, USA), anti-acetylated lysine (Aviva Systems Biology, CA), anti-p300 (Santa Cruz Biotechnology, USA), anti-ER-α (ImmunoWay Biotechnology Company), anti-HSC70 (Proteintech), anti-Ki67 (Santa Cruz Biotechnology), and IL-6 neutralizing antibody (Abcam). Trichostatin A (TSA) and cycloheximide (CHX) were separately purchased from Beyotime Biotechnology (China) and MedChem Express (USA). 4OH-TAM and ASA were purchased from Sigma-Aldrich.

Whole cell lysates from PrECs, CWR22Rv1-Cas9, CWR22Rv1-LV-MEIS1, LAPC4-Cas9 and LAPC4-LV-MEIS1 cells were prepared in protein lysis buffer (20 mM Tris, pH 7.5; 150 mM NaCl; 1 mM EDTA; 1 mM EGTA; 2.5 mM sodium pyrophosphate; 1 mM sodium glycerophosphate; 1 mM sodium orthovanadate; 1% Triton-X 100) supplemented with cOmplete Mini Protease Inhibitor Cocktail (#11873580001, Sigma-Aldrich). To pull down MEIS1, 1000 µg of precleared cell lysates were incubated with 2 μg of Anti-HOXB13 (EPR17371; Cat #ab201682, Abcam) antibody overnight at 4°C. The lysates were then incubated with immobilized Protein A/G Agarose beads (#20421, Thermo Scientific, USA) overnight at 4°C. Finally, the beads were washed three time with protein lysis buffer and centrifuged at 600 g for 5 min. Co-immunoprecipitated proteins were then eluted from the beads by adding 40 μL of RIPA buffer to 5 × SDS PAGE sample buffer and heating at 95°C for 5 min. Samples were further size-fractionated on 10% SDS-polyacrylamide gels. The resolved gels were electro-transferred onto nitrocellulose membranes and probed for Anti-MEIS1 (1:1000, Cat #0T12A3, Origene) and Anti-HOXB13 (1:1000) with respective secondary antibodies (1:10000). The membranes were scanned on Odyssey imaging system (LI-COR Biosciences) and analyzed with LI-COR Image Studio software.