Pabpc1

Protein samples were prepared in reduced and denatured forms (32 (link)) and resolved using SDS-PAGE. The proteins were transferred to a nitrocellulose membrane and then blocked with 5% nonfat milk in TBS-T (0.02 M Tris–HCl, 0.16 M NaCl, and 0.1% Tween-20, pH 7.4), at room temperature, for 1 h. The membranes were incubated overnight with primary antibodies PABPC4 (Bethyl, #A301-466A), NCoR1 (Affinity, #AF0270), PABPC1 (Thermo Fisher Scientific, #PA5-29883), FLAG (Sigma-Aldrich, #F1804), α-tubulin (Sigma-Aldrich, #T9026), OXPHOS (proteins of mitochondrial ETC) (Abcam, #ab110413), PPARD (Thermo Fisher Scientific, #PA1-823A), eIF4G (Cell Signaling Technology, #2498), puromycin (Merck, #MABE343), Vinculin (Cell Signaling Technology, #4650), Lamin A (Santa Cruz Biotechnology, #sc-71481), and β actin (Santa Cruz Biotechnology, #81178), Akt (Cell Signaling Technology, #9272), p-Akt^Thr308 (Cell Signaling Technology, #9275), ubiquitin (Abcam, #ab7254), and GST (Sigma-Aldrich, #G7781). The membrane was then washed with TBS-T and incubated with horseradish peroxidase–conjugated secondary antibody (1:10,000) in TBS-T solution containing 5% nonfat for 1 h. Membranes were washed with TBS-T and then added the peroxidase substrate SuperSignal West Plus (Thermo Fisher Scientific), and the band intensities were captured in the ImageQuant LAS500 (GE Healthcare).

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).

Immunoblots were performed following standard protocols (www.cshprotocols.org). HEL cells were lysed in a modified RIPA buffer (150 mM NaCl, 50 mM Tris, pH 7.5, 2 mM MgCl₂, 0.1% SDS, 2 mM DDT, 0.4% Triton X-100), 1× complete protease inhibitor cocktail (complete Mini EDTA-free, Roche) on ice for 15 min. Cell lysates were quantified using the Pierce BCA Protein Assay Kit (ThermoFisher). The Trans-Blot Turbo transfer system was used according to the manufacturer’s instructions. The following commercial antibodies were used: CXXC1 (Cell Signal, 1:250), PABPC4 (Novus Biologicals, 1:500), PABPC1 (ThermoFisher, 1:500), WTAP (Abcam, 1:500), BRD7 (ThermoFisher, 1:250), STK40 (ThermoFisher, 1:250), TADA2B (Abnova, 1:250), Beta-actin (Cell Signaling, 1:1000), and Beta-tubulin (Abcam, 1:1000). An Odyssey infrared imaging system (LI-COR) was used to visualize blots following the manufacturer’s instructions. The Image Studio software was used to semi-quantify the blots.
The puromycin incorporation assay was performed as previously reported^{92 (link)}. HEL cells were transduced with sgNTC or sgWTAP and cultured for 10 day. On day 10, the cells were treated with 10 µg/mL of puromycin for 10 min at 37 °C. Immunoblotting for puromycin incorporation (Millipore Sigma, 1:1000) was done as described above.