Amersham imager 600 qc

See Supplementary Table 4 for a complete list of antibodies and dilutions used. Ba/F3 cells were lysed in Cell Extraction Buffer (Sample Diluent Concentrate 2, Bio-Techne), and patient-derived cells were lysed in radioimmunoprecipitation buffer; lysis buffers were supplemented with phosphatase (PhosSTOP) and protease inhibitors (cOmplete Mini Protease Inhibitor Cocktail), both obtained from Merck. Total cellular proteins (10 µg for Ba/F3 cells, 20 µg for Ba/F3 xenografts or 25 µg for other cells) were subjected to SDS–PAGE. After electrophoresis, the separated proteins were transferred to PVDF membranes (Bio-Rad Laboratories), and then membranes were blocked in Blocking One-P (Nacalai Tesque), before incubation overnight with primary antibodies on a shaker in a cold room. The next day, membranes were washed and then soaked with HRP-linked anti-rabbit IgG (Cell Signaling Technology). The bands of the target proteins were detected with SuperSignal West Dura Extended Duration Substrate (Thermo Fisher Scientific) by the Amersham Imager 600 QC (Cytiva) or exposed to X-ray film and visualized using a Kodak X-ray developer.

See Supplementary Table 4 for a complete list of antibodies and dilutions used. Ba/F3 cells were lysed in Cell Extraction Buffer (Sample Diluent Concentrate 2, Bio-Techne), and patient-derived cells were lysed in radioimmunoprecipitation buffer; lysis buffers were supplemented with phosphatase (PhosSTOP) and protease inhibitors (cOmplete Mini Protease Inhibitor Cocktail), both obtained from Merck. Total cellular proteins (10 µg for Ba/F3 cells, 20 µg for Ba/F3 xenografts or 25 µg for other cells) were subjected to SDS–PAGE. After electrophoresis, the separated proteins were transferred to PVDF membranes (Bio-Rad Laboratories), and then membranes were blocked in Blocking One-P (Nacalai Tesque), before incubation overnight with primary antibodies on a shaker in a cold room. The next day, membranes were washed and then soaked with HRP-linked anti-rabbit IgG (Cell Signaling Technology). The bands of the target proteins were detected with SuperSignal West Dura Extended Duration Substrate (Thermo Fisher Scientific) by the Amersham Imager 600 QC (Cytiva) or exposed to X-ray film and visualized using a Kodak X-ray developer.

See Supplementary Table 4 for a complete list of antibodies and dilutions used. Ba/F3 cells were lysed in Cell Extraction Buffer (Sample Diluent Concentrate 2, Bio-Techne), and patient-derived cells were lysed in radioimmunoprecipitation buffer; lysis buffers were supplemented with phosphatase (PhosSTOP) and protease inhibitors (cOmplete Mini Protease Inhibitor Cocktail), both obtained from Merck. Total cellular proteins (10 µg for Ba/F3 cells, 20 µg for Ba/F3 xenografts or 25 µg for other cells) were subjected to SDS–PAGE. After electrophoresis, the separated proteins were transferred to PVDF membranes (Bio-Rad Laboratories), and then membranes were blocked in Blocking One-P (Nacalai Tesque), before incubation overnight with primary antibodies on a shaker in a cold room. The next day, membranes were washed and then soaked with HRP-linked anti-rabbit IgG (Cell Signaling Technology). The bands of the target proteins were detected with SuperSignal West Dura Extended Duration Substrate (Thermo Fisher Scientific) by the Amersham Imager 600 QC (Cytiva) or exposed to X-ray film and visualized using a Kodak X-ray developer.