Figure 2. Efficient reversion of gene trap mutations and Floxin-mediated engineering of new alleles.

(a) Plot of β-galactosidase activity in cell lines of the indicated genotypes. Error bars are standard deviations from 1–4 different experiments with a minimum of 3 replicates each. (b) Immunoblot showing Suz12 (left and middle panels, 20 µg protein per lane) and Sall4 (right panel, immunoprecipitated from 500 µg total protein for each cell line) protein levels in the cell lines of the indicated genotypes. (c) Immunoblots showing expression of full length Myc-tagged Ofd1 (left panel, 25 µg protein per lane; right panel, immunoprecipitate from 800 µg total protein for each cell line), and of Ofd1 in Ofd1^Rev cells (middle panel, immunoprecipitate from 4 mg total protein for each cell line). (d, e) Representative fluorescence micrographs of cell lines of the indicated genotypes. Cilia (acetylated Tubulin, green), centrosomes (γ-Tubulin, red), DNA (DAPI, blue). (f) Representative fluorescence micrograph of Ofd1-Myc localization. Ofd1 (Myc, green), centrosome (Centrin, red), DNA (DAPI, blue). (g) Representative fluorescence micrograph of Suz12-TAP localization. Suz12 (Flag, green), centrosome (Acetylated Tubulin, red), DNA (DAPI, blue). (h) Immunoblot showing wild type Suz12 and Suz12-TAP protein downregulation upon differentiation of Suz12^Suz12TAP/+ cells. 3 µg protein per lane. Scale bars 5 µm, with magnified inset scale bar 1 µm.