Journal: eLife
Article Title: Rapid DNA replication origin licensing protects stem cell pluripotency
doi: 10.7554/eLife.30473
Figure Lengend Snippet: ( a ) Chromatin flow cytometry of RPE cells stained for loaded MCM2 (anti-MCM2), loaded MCM3 (anti-MCM3), and DNA content (DAPI). All three plots are the same sample. Cells stained with monoclonal MCM2 antibody for loaded MCM2 co-stain equally well for a polyclonal MCM3 antibody for loaded MCM3. ( b ) Chromatin flow cytometry of RPE cells treated with 100 nM siControl or a 100 nM pool of siMCM3 for 72 hr, measuring DNA content (DAPI), DNA synthesis (EdU incorporation), and loaded MCM3 (anti-MCM3). Loaded MCM3 levels decrease with siMCM3 treatment, demonstrating antibody specificity. ( c ) Immunoblot of cells from ( b ). ( d ) RPE cells synchronized in G0 by contact inhibition. Left: Total protein flow cytometry of G0 cells measuring total MCM (anti-MCM2), DNA synthesis (EdU incorporation) and DNA content (DAPI). Right: Chromatin flow cytometry of G0 cells measuring loaded MCM (anti-MCM2), DNA synthesis (EdU incorporation) and DNA content (DAPI). G0 cells lack loaded MCM, demonstrating antibody specificity. ( e ) Immunoblot of cells for ( d ) demonstrating G0 synchronization. ( f ) Immunoblots of cells fractionated into soluble protein and pellet (chromatin associated protein) using a normal-salt CSK buffer (100 mM NaCl) or high-salt CSK buffer (300 mM NaCl). High salt 300 mM NaCl strips away weakly chromatin-associated proteins, including Cdc6. ( g ) Chromatin flow cytometry of cells lysed with normal 100 mM NaCl CSK or high salt 300 mM NaCl CSK. ( h ) Histograms of only the G1-MCM DNA -positive cells from ( g ). The loaded MCM in G1 remains constant with high salt, demonstrating the chromatin flow cytometry measures DNA-loaded MCM. Note the harsh 300 mM salt alters nuclear morphology and scatter by flow cytometry; for this reason, we used the 100 mM NaCl extraction for all other flow cytometry experiments.
Article Snippet: Antibody , Anti-Orc1, rabbit polyclonal , Bethyl Laboratories , Cat#A301-892A; AB_1524103 , 1:1000 (IB).
Techniques: Flow Cytometry, Staining, DNA Synthesis, Western Blot, Inhibition, Extraction
Bethyl is a verified supplier 
