Transcription Factor Knockdown Protocol

Knockdown of TAL1, RUNX1 and GATA1 was achieved with shRNAs present in the pGIPZ vector (Open Biosystems) [17 (link)]. The accession numbers of the pGIPZ constructs is given in Supplementary Material. Controls were expressing non-targeting shRNA from the corresponding backbone. Knockdown was verified by qRT-PCR (Figure 5). A corresponding western blot from the same lysates showing the decreased protein amount of TAL1, RUNX1 and GATA1 is shown in [17 (link)]. For western blot analysis whole cell extracts from transfected HEK293 cells were used. Proteins were separated using SDS-PAGE and transferred to PVDF membranes. Membranes were blocked with Roti-Block (Carl Roth) and exposed to primary antibody dilution over night at 4°C. Afterwards membranes were incubated with appropriate secondary antibodies coupled to horseradish peroxidase (HRP) for 1h at room temperature. Signals were visualized using Western Super ECL reagent (Pierce) and analysed using X-ray film. Immunoblotting analysis was performed using anti-TAL1 (sc-12984, Santa Cruz), anti-RUNX1 (ab23980, Abcam), anti-GATA1 (sc-1233, Santa Cruz) and anti-Tubulin (ab7291, Abcam).