Flow cytometry–sorted GMPs (see above) from leukemic Lp30 and WT mice, corresponding to approximately 10,000 cells for each sample, were boiled in SDS-loading buffer for 5 min, subjected to nucleic acid degradation by Benzonase (E1014-5KU, Sigma-Aldrich) for 20 min on ice, and spun for 20 min at 20,000g. Material was size-separated using NuPAGE precast 4 to 12% Bis-tris gels (Invitrogen). The Cell Signaling protocol (www.cellsignal.com/support/protocols/western.html) was used for blotting. ImageJ was used for quantifications using program guidelines (http://rsb.info.nih.gov/ij/). CEBPA Ab [at 1:1000 dilution in 5% (w/v) BSA, 1 hour at room temperature] corresponds to the one used for ChIP (clone 14AA, sc-61, Santa Cruz Biotechnology), and loading control was anti-Histone H3 [1:5000 in 5% (w/v) nonfat milk blocking buffer, 2 hours at room temperature; Ab10799, Abcam]. A high-sensitivity chemiluminescence HRP detection kit was used (Amersham ECL Prime Western Blotting Detection Reagent, RPN2232).

Note: The content above has been extracted from a research article, so it may not display correctly.



Q&A
Please log in to submit your questions online.
Your question will be posted on the Bio-101 website. We will send your questions to the authors of this protocol and Bio-protocol community members who are experienced with this method. you will be informed using the email address associated with your Bio-protocol account.



We use cookies on this site to enhance your user experience. By using our website, you are agreeing to allow the storage of cookies on your computer.