Ab196026

Assays were performed as described previously.²⁵ For immunohistochemistry, all specimen sections were incubated with anti‐TPO rabbit monoclonal antibodies (1:100; ab196026; Abcam) overnight at 4°C. Staining intensity represented TPO expression and was scored as follows: 0 (no staining), 1 (weak), 2 (moderate) or 3 (high). Percentage scores were assigned as follows: 1 (1%‐25%), 2 (26%‐50%), 3 (51%‐75%) and 4 (76%‐100%). The two scores were multiplied for each specimen to give a final score of 0‐12. Specimens with scores >6 were considered TPO‐positive, whilst those with scores ≤ 6 were considered TPO‐negative. For immunofluorescence, cells were incubated with anti‐TPO rabbit monoclonal antibodies (1:50; ab196026; Abcam) or anti‐EGFR rabbit monoclonal antibodies (1:50; #4267; Cell Signaling Technology) overnight at 4°C. Images were acquired with an Olympus FV3000 confocal laser scanning microscope (Tokyo).

KG1a cells were lysed by the non-denaturing lysis buffer. Next, the supernatant of cell lysis was pre-cleaned with protein A/G magnetic beads (Thermo Fisher Scientific) for 2 h at 4 °C. Subsequently, about 300 μg of protein were incubated with 1μg G-CSF antibody (#ab181053, Abcam) or EP300 antibody (#ab275378, Abcam) and 25 microliters of protein A/G magnetic beads for immunoprecipitation at 4 °C overnight. Following the incubation with antibody and protein A/G magnetic beads, protein A/G magnetic beads were collected using magnetic separation device (Thermo Fisher Scientific), and precipitated complexes were cleansed by washing buffer (Thermo Fisher Scientific). Finally, bound proteins were analyzed by WB using anti-ELANE (1:500, # bs-6982R, Bioss) or anti-TPO (1:500, # ab196026, Abcam). Rabbit IgG was used for negative control.