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. 2018 Mar 1;145(5):dev149419. doi: 10.1242/dev.149419

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© 2018. Published by The Company of Biologists Ltd

This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution and reproduction in any medium provided that the original work is properly attributed.

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Fig. 4. — Cbfb^−/− have reduced RUNX1 protein levels and cannot initiate EHT. (A) Western blot for CBFβ on CRISPR/Cas9-generated Cbfb^−/− mESC clones. Lane 3 shows a wild-type (wt) mESC positive control. (B) Representative flow cytometry plots for Cbfb^−/− day 2 and day 7 EHT cultures. (C-E) Four Cbfb^−/− clones were differentiated across two independent experiments. Individual clones and mean are shown. (C,D) Flow cytometry data for four Cbfb^−/− clones analyzed on day 2-4 (C) and day 7 (D) of EHT culture. (E) Time-course qPCR analysis of Cbfb^−/− EHT cultures. Data are normalized to the wild-type line. (F) Time-course western blot analysis of EHT cultures for RUNX1, CBFβ and β-actin on two Cbfb^−/− clones (lanes 2, 5, 8, and 3, 6, 9) and wild type (lanes 1, 4, 7). β-actin-normalized relative densities for RUNX1 and CBFβ are shown. (G,H) Flow cytometry analysis of four Cbfb^−/− clones across five independent mast cell differentiations.