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. 2016 Aug 18;35(19):2152–2166. doi: 10.15252/embj.201694462

Figure EV5. Generation of RPE‐1 cells expressing endogenously tagged Sas‐6‐EGFP.

Figure EV5

  1. Scheme of WT SASS6 locus and the targeted SASS6 allele obtained by rAAV‐mediated homologous recombination. Black rectangles represent exons and black lines introns. After insertion of the EGFP coding sequence and the G418 resistance cassette, cells were selected by addition of G418 in the medium. Then, the neomycin cassette was removed using an adenovirus‐expressed Cre recombinase (Vector Biolabs, Malvern, PA, USA), and clones were isolated by serial dilution (loss of resistance to G418 was confirmed by replica plating). Finally, positive cells were confirmed by fluorescence microscopy.
  2. Western blot analysis of parental RPE‐1 and Sas‐6‐EGFP‐expressing RPE‐1 cells. Protein extracts were probed with anti‐Sas‐6 (left panel) and anti‐GFP (right panel) antibodies. Histogram shows quantification of the numbered bands. Results presented are means ± SEM. Band intensity for WT alleles was set to 100%.
  3. Maximum projection fluorescence microscopy image showing asynchronously growing RPE‐1 cells expressing Sas‐6‐EGFP. Scale bar: 10 μm. The inset shows an enlargement of the boxed interphase centrosome.
  4. Efficacy of cell cycle synchronization was demonstrated by subjecting cell lysates to Western blotting with the indicated antibodies against cell cycle markers.