Figure 1

Generation of BAZ1B knockout cells. (a) Schematic representation of the BAZ1B gene. Exons: numbered vertical bars. Introns: connecting lines. Exon 7 is bracketed by the blue lines, and expanded beneath. The targeting construct is shown beneath, indicating the location of the neomycin resistance gene and the extent of the homology arms (regions between the vertical dotted lines). Left and right side screening PCRs are indicated below by inward-facing arrowheads. The NHEJ PCR is shown above Exon 7, and the ZFN cutting site is indicated by the white arrowheads within the exon. (b) Example of an ethidium bromide stained gel showing a positive clone by PCR. (c) DNA sequence alignment of parental RPE1 with NHEJ disrupted clones. The cutting site for the ZFNs is highlighted in red. Deletions in NHEJ clones are indicated by the red gaps; insertions by the blue triangle. (d) qRT-PCR analysis of BAZ1B showing levels in a heterozygous mutant (BAZ1B +/-), a clone with one allele targeted and one disrupted by NHEJ (BAZ1B -/NHEJ) and a clone targeted at both alleles (BAZ1B -/-) relative to parental RPE1. (e) Indirect immunofluorescence showing the distribution of WSTF in RPE1, a BAZ1B +/-, BAZ1B -/NHEJ and a BAZ1B -/- clone. WSTF is shown in green for the Bethyl Labs anti-WSTF antibody and red for the Cell Signaling anti-WSTF antibody. The white arrowheads indicate the location of the Xi. The nucleus is counterstained with DAPI (blue). (f) Western blot for WSTF with the Cell Signaling antibody in RPE1, a BAZ1B +/-, and various independent BAZ1B knockout mutants. Actin is shown immediately below. (g) Schematic map of WSTF showing the relative location of functional domains, and the truncated proteins possible in the different mutant types. (h) Western blot using the Bethyl Labs anti-WSTF antibody as described for part-f above.