Fig. 4
Interaction candidates are coexpressed with TBX2 in the pulmonary mesenchyme and interact in HEK293 cells. a Co-immunofluorescence analysis of candidate interaction partners (red) and TBX2 (green) on frontal sections of the right lung of E14.5 Tbx2cre/+ embryos. Antigens are color-coded and nuclei were counterstained with DAPI (blue). Insets or selected regions in overview images are magnified in rows 2,4 and 6. b In situ proximity ligation assay of TBX2 and candidate interaction partners on 10 µm frontal sections of E14.5 wildtype and Tbx2cre/fl mutant lungs. Direct interaction is visualized by small red fluorescent dots. Larger more diffuse orange stains are due to auto-fluorescence of blood cells. Nuclei are counterstained with DAPI (blue). c Western blot analysis of co-immunoprecipitation experiments for verification of TBX2 interaction with candidate proteins on 10% SDS polyacrylamide gels. Detection was performed with an anti-TBX2 primary antibody and developed with chemoluminescence-IHC. Arrows indicate TBX2 bands. Lanes were loaded as follows: No antibody: IP without specific antibody resembling negative IP-control; 5% input: 5% of crude cell extract before precipitation; empty: no protein loaded; IP: co-immunoprecipitate with antibody for specific candidate. Expected molecular weight for TBX2.HA approx. 76.2 kDa