Analyzing WTAP Expression in MEP Progenitors

Adult bone marrow CD34+ HSPCs were transduced with shScr or shWTAP lentivirus and cultured in the expansion growth factor cocktail as described above. Five days post transduction the cells were flow sorted for the transduced (GFP+) MEP progenitor population as shown in Fig. 6g. The sorted cells were then immediately loaded onto the Milo small scWest Chip (ProteinSimple) following the manufactures instructions^{93 (link)}. The following run conditions were used: lysis—5 s; electrophoresis—70 s; UV capture 4 min. The chips were probed with the following antibodies: PABPC4 (Novus Biologicals, 1:20), PABPC1 (ThermoFisher, 1:20), WTAP (Abcam, 1:20), Beta-tubulin (Abcam, 1:20) and Alexa Fluor 555 donkey anti-rabbit (Invitrogen, 1:40) was used for the secondary antibody. Chips were scanned on a GenePix 4000B (Molecular Devices) and analyzed with the scout software package (ProteinSimple).