phh3 s10  (Cell Signaling Technology Inc)

Journal: BMC Cell Biology

Article Title: Cytometry of chromatin bound Mcm6 and PCNA identifies two states in G1 that are separated functionally by the G1 restriction point 1

doi: 10.1186/1471-2121-11-26

Figure Lengend Snippet: Effect of cell cycle inhibitors . Exponentially growing BJ1 cells were switched to medium with 0.03% FBS and inhibitors. Controls (no switch or addition) and treated cells were extracted with TX-100, fixed with MeOH. Controls were processed at time zero; treated samples were processed at 6 or 8 hours. Samples were then stained for Mcm6, PCNA, phospho-S10-histone H3, and DNA and subjected to cytometry as described in the Methods section. The percentages of G1p+eS, eS, G1a were normalized to the same fraction in the control sample. Normalized eS (early S phase), G1p+eS (high-Mcm6 cells+ early S phase), and G1a (low-Mcm6 G1 cells) are plotted for cells treated with mimosine ( A ), aphidicolin ( B ), and nocodazole ( C ). Serum withdrawal stopped forward movement of cells from the G1a compartment but not G1p or S (middle bars). Serum withdrawal plus mimosine prevented cells from entering or exiting the G1p compartment ( A ). Serum withdrawal plus aphidicolin arrested cells in S ( B ). The frequency of eS increased over 6 hours as cells moved from G1p to eS. Serum withdrawal plus nocodazole prevented cells from entering the G1a compartment ( C ).

Article Snippet: The following antibodies were used for cytometry and immunoblotting: Phycoerythrin-conjugated and unconjugated anti-Mcm6 (PE-Mcm6), clone K1-Mcm6, (BD Biosciences); fluorescein isothiocyanate-conjugated and unconjugated anti-PCNA (FITC-PCNA), clone PC10 (BD Biosciences); BM28 (anti-Mcm2), clone 46, (BD Biosciences); anti-Cdc6, clone DCS-180, (Upstate, Lake Placid, NY); anti-ORC3, clone 1D6, (Upstate); Alexa Fluor 647-conjugated anti-phospho-S10-histone H3 (A647-pHH3) (Cell Signaling, Danvers, MA); FITC-conjugated goat anti-mouse F(ab) 2 (BD Biosciences).

Techniques: Staining, Cytometry