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. 2015 Sep 29;4:e10874. doi: 10.7554/eLife.10874

Figure 4. Runx1 and CBFβ are co-expressed and form a complex in DRG neurons.

(A and B) Double immunostaining of Flag and Runx1 in wildtype and CbfbFlag/+ DRGs at P0 confirms the specificity of the Flag antibody. Note that Flag immunoreactivity in wildtype DRGs is nearly undetectable. (C–E) Double immunostaining of Flag and Runx1 in CbfbFlag/+ DRGs at P0 shows extensive colocalization between Flag-CBFβ and Runx1 (arrows). Note that CBFβ is expressed in many more DRG neurons than those that are Runx1+ . See more examples in Figure 4—figure supplement 1E–G. (F) Co-immunoprecipitation experiments using a Flag antibody for immunoprecipitation from DRGs lysates from P0 wildtype and CbfbFlag/Flag animals. Subsequent detection with Runx1 and CBFβ antibodies shows that Runx1 associates with Flag-CBFβ from DRGs of CbfbFlag/Flag animals, but not wildtype controls, indicating the formation of a Runx1/CBFβ complex in the DRG. Scale bar, 50 μm.

DOI:http://dx.doi.org/10.7554/eLife.10874.009

Figure 4.

Figure 4—figure supplement 1. Generation of the CbfbFlag allele and more detailed characterization of the temporal and spatial patterns of Cbfb expression.

Figure 4—figure supplement 1.

(A) Schematic showing the targeting strategy used for generation of CbfbFlagknockin mice. Following germ-line transmission, the Neo selection cassette was removed by crossing the carrier to a mouse expressing FlpE recombinase in the germ line. A Bstz171 restriction site was introduced immediately downstream of the Flag sequence to facilitate Southern screening of embryonic stem (ES) cells. Flag epitope sequences, LoxP and FRT sites are shown as red filled triangles, open and filled triangles respectively. (B–D) In situ hybridization analysis of Cbfb expression in wildtype DRGs at E13.5, E16.5 and P14 reveals a widespread pattern of expression throughout development. Note that Cbfb is expressed at varying levels in DRG neurons. (E–G) Double immunostaining of Flag (green) and Runx1 (red) in CbfbFlag/ + DRGs at E13.5, E16.5 and P14 shows a pattern of CBFβ protein similar to its mRNA distribution.
DOI:http://dx.doi.org/10.7554/eLife.10874.010