Figure 2. A stable HeLa H2B-2FP cell... | Rockefeller University Press

Figure 2.

$Figure 2. A stable HeLaH2B-2FP cell line coexpressing H2B-EGFP and mCherry-H2B histones. (A) Fluorescent-tagged H2B histones in HeLaH2B-2FP cells imaged by wide-field fluorescence microscopy in interphase (top) and metaphase spreads (bottom). Insets show high magnification views. Arrowheads indicate concentrated heterochromatin in the chromosome, which was brightly stained with DAPI. (B) High resolution imaging of HeLaH2B-2FP cells at different stages of mitosis. (C) Total cell lysates from HeLa and HeLaH2B-2FP cells analyzed by PAGE and Western blotting with antibodies against H2B (lanes 3 and 4), GFP (lanes 5 and 6), or mCherry (lanes 7 and 8). (D) Salt extraction of histones from HeLaH2B-2FP cells. Proteins either from whole cells (total; lane 1) or the supernatant fractions from 0, 0.5, and 1 M NaCl salt extractions of chromatin (sup; lanes 2–4) were separated on SDS-PAGE gels and either stained with Coomassie blue (top) or blotted and probed with the indicated antibodies. (E) FACS analyses showing cell cycle distribution of HeLaEGFP, HeLaH2B-GFP, and HeLaH2B-2FP stable cell lines. Bars, 10 µm.$

A stable HeLa^H2B-2FP cell line coexpressing H2B-EGFP and mCherry-H2B histones. (A) Fluorescent-tagged H2B histones in HeLa^H2B-2FP cells imaged by wide-field fluorescence microscopy in interphase (top) and metaphase spreads (bottom). Insets show high magnification views. Arrowheads indicate concentrated heterochromatin in the chromosome, which was brightly stained with DAPI. (B) High resolution imaging of HeLa^H2B-2FP cells at different stages of mitosis. (C) Total cell lysates from HeLa and HeLa^H2B-2FP cells analyzed by PAGE and Western blotting with antibodies against H2B (lanes 3 and 4), GFP (lanes 5 and 6), or mCherry (lanes 7 and 8). (D) Salt extraction of histones from HeLa^H2B-2FP cells. Proteins either from whole cells (total; lane 1) or the supernatant fractions from 0, 0.5, and 1 M NaCl salt extractions of chromatin (sup; lanes 2–4) were separated on SDS-PAGE gels and either stained with Coomassie blue (top) or blotted and probed with the indicated antibodies. (E) FACS analyses showing cell cycle distribution of HeLa^EGFP, HeLa^H2B-GFP, and HeLa^H2B-2FP stable cell lines. Bars, 10 µm.