Figure 1. Fluorescently labeled Fab... | Rockefeller University Press

Figure 1.

$Figure 1. Fluorescently labeled Fab binds to phosphorylated histone H3S10 during mitosis in HeLa cells. (A) Specificity of mAbs evaluated by ELISA using the indicated peptides. The peptides that reacted with individual mAbs are indicated in the graphs. (B) Immunofluorescence. Fixed cells were stained with Fab311-488 (Alexa Fluor 488–labeled Fab from CMA311; red), Fab313-555 (Alexa Fluor 555–labeled Fab from CMA313; green), and anti–CENP-C (blue). Images of a prophase cell are shown with magnified views of boxed area. See Fig. S1 for other cells at different phases of the cell cycle. (C) Time-lapse images of a cell loaded with Fab311-488. Arrows indicate concentrations of Fab311-488. See Video 1. (D) Period of mitosis in Fab311-488–loaded and control cells. By collecting three z-stack images every 3 min, the period from prophase to anaphase was measured (n = 27) using phase-contrast images. No significant difference was seen; P = 0.66 (Student’s t test). (E) The mobility of Fab311-488 by FRAP. After bleaching a 2-µm spot (white circle), images were collected for 12 s every 0.13 s (left) or for 120 s every 0.4 s (right). Means ± SD are shown (n = 12). The red line shows the fitted curve using single exponential association kinetics. Residence time (k−1; k = association coefficient) of the transiently bound fraction is calculated as 44 s. (F) H3S10ph detected in live and fixed samples. Cells loaded with Fab311-488 were imaged every 3 min. When foci were detected in two consecutive frames (01:00 and 01:03), cells were fixed and immunolabeled with Fab311-555. Foci observed during live imaging (arrows) were also detected after fixation and immunolabeling. Bars: (B, C, and F) 5 µm; (E) 1 µm.$

Fluorescently labeled Fab binds to phosphorylated histone H3S10 during mitosis in HeLa cells. (A) Specificity of mAbs evaluated by ELISA using the indicated peptides. The peptides that reacted with individual mAbs are indicated in the graphs. (B) Immunofluorescence. Fixed cells were stained with Fab311-488 (Alexa Fluor 488–labeled Fab from CMA311; red), Fab313-555 (Alexa Fluor 555–labeled Fab from CMA313; green), and anti–CENP-C (blue). Images of a prophase cell are shown with magnified views of boxed area. See Fig. S1 for other cells at different phases of the cell cycle. (C) Time-lapse images of a cell loaded with Fab311-488. Arrows indicate concentrations of Fab311-488. See Video 1. (D) Period of mitosis in Fab311-488–loaded and control cells. By collecting three z-stack images every 3 min, the period from prophase to anaphase was measured (n = 27) using phase-contrast images. No significant difference was seen; P = 0.66 (Student’s t test). (E) The mobility of Fab311-488 by FRAP. After bleaching a 2-µm spot (white circle), images were collected for 12 s every 0.13 s (left) or for 120 s every 0.4 s (right). Means ± SD are shown (n = 12). The red line shows the fitted curve using single exponential association kinetics. Residence time (k⁻¹; k = association coefficient) of the transiently bound fraction is calculated as 44 s. (F) H3S10ph detected in live and fixed samples. Cells loaded with Fab311-488 were imaged every 3 min. When foci were detected in two consecutive frames (01:00 and 01:03), cells were fixed and immunolabeled with Fab311-555. Foci observed during live imaging (arrows) were also detected after fixation and immunolabeling. Bars: (B, C, and F) 5 µm; (E) 1 µm.