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Figure 2. Time-Course Analysis at the Single-Nucleus Level Reveals the Temporal Recruitment of Oocyte B4 and of Pol II Phosphorylation(A) Experimental set-up to examine binding of oocyte factors to transplanted nuclei.(B) Proportion of transplanted nuclei stained by B4, hypophosphorylated Pol IIA, Ser5P Pol II, and Ser2P Pol II at different times after NT. n = number of nuclei scored.(C and D) B4 (green), Pol IIA (red), Ser5P Pol II (blue), and Ser2P Pol II (magenta) immunofluorescence detection at the indicated times after NT of MEF nuclei. DAPI is in white. Scale bars in DAPI, 10 μm. Images represent projected Z sections (C–F).(E) Immunofluorescence detection of B4 (green) and total Pol II (red) 6 hr after nuclear transfer of MEF nuclei. Total Pol II antibody recognizes hypophosphorylated Pol IIA, Ser5P Pol II and Ser2P Pol II. Arrows indicate nuclei that are positive for B4 but negative for total Pol II. DAPI is in blue.(F) Immunofluorescence detection of B4 (green), Pol IIA (red), and Ser5P Pol II (blue). Arrowheads indicate nuclei that are positive for B4 and Pol IIA but negative for Ser5P Pol II. Arrows show nuclei positive for all markers. (G) Proportion of transplanted nuclei stained with B4, Pol IIA, Ser5P Pol II, or Ser2P Pol II, which are also stained with B4 (green), Pol IIA (orange), Ser5P Pol II (blue), or Ser2P Pol II (magenta) at 6 hr.See also Figure S2 and Table S3. Image published in: Jullien J et al. (2014) Image downloaded from an Open Access article in PubMed Central. © 2014 The Authors. The Journal of Physiology published by John Wiley & Sons Ltd on behalf of The Physiological Society Larger Image Printer Friendly View

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