Western Blot for Hematopoietic Stem Cells

WB was performed as described previously^{38 (link)}. Approximately 20,000 SLAM HSCs were sorted into 250 μl PBS containing 20% trichoracetic acid (TCA). The concentration of TCA was adjusted to 10% after sorting. Extracts were incubated on ice for 30 min and centrifuged at 13,000 rpm for 10 min at 4°C. Precipitates were washed in pure acetone (Fisher scientific, A18-4) twice and the dried pellets were solubilized in 9 M urea, 2% Triton X-100, and 1% DTT together with 1X LDS buffer (Invitorgen, NP0007). Samples were separated on NuPAGE 4–12% Bis-Tris protein gels (Invitrogen, NP0336BOX) and transferred to PVDF membrane (Millipore). The blots were incubated with primary antibodies overnight at 4°C and then with secondary antibodies. NativePAGE™ 4-16% Bis-Tris protein gel was used to detect MPL aggregates in HSCs as manufacturer’s instructions^{39 (link)} (Invitrogen, BN1002BOX). Blots were developed with the SuperSignal West Femto chemiluminescence kit (Thermo Scientific, 34096). Antibodies and reagents used are in Supplementary Table 1 and 2.