Journal: BMC Genomics

Article Title: Fluorescence activated cell sorting followed by small RNA sequencing reveals stable microRNA expression during cell cycle progression

doi: 10.1186/s12864-016-2747-6

Figure Lengend Snippet: Fluorescence activated cell sorting (FACS) analysis of stained cells and validation of cell cycle sorted populations in HDFa, NCI-H295R and HeLa cells. Panels a - c : FACS analysis shows cell cycle distribution of stained cells before (first image) and after sorting to G1, S and G2 phases (second, third and fourth images, respectively; Panel a : HDFa, Panel b : NCI-H295R, Panel c : HeLa. Intervals of fluorescence intensity (shown as vertical bands) were defined to gate G1, S and G2 phases, respectively. Panels d-e : Western blot analysis confirms the high efficacy of cell cycle sort (Panel d : NCI-H295R, Panel e : HeLa). Density values of phospho(Tyr15)-CDC-2 were first normalized to β-actin loading control and were further normalized to G1 phase (displayed above each band)

Article Snippet: Membranes were blocked with 5 % non-fat dry milk in TBS for 60 min at room temperature, and were incubated with primary phospho-CDC-2 (Tyr15) antibody (Cell Signaling Technology, cat. No.: 9111, dilution: 1:500) at 4 °C for 16 h. Thereafter, membranes were washed 5 times with 0.05 % Tween-20 containing TBS, and were incubated with secondary antibody (Cell Signaling Technology, cat. No.: 7074, dilution: 1:2000).

Techniques: Fluorescence, FACS, Staining, Western Blot