Figure 6: Nr2f2 interaction with Fog2.

Co-immunoprecipitation analysis of Nr2f2 and Fog2 association in NRK52E cells. Extracts from NRK52E cells were transfected with expression plasmid for Nr2f2^mutant and Nr2f2^wildtype. (a) Western blotting (WB) of cell lysates for Nr2f2 protein to confirm the overexpression of both forms of Nr2f2. Lanes labelled from the left—control: NRK52E cell lysate; empty vector: NRK52E cells transfected with empty pcDNA 3.1(-B) myc/His vector; Nr2f2^wt: NRK52E cells transfected with Nr2f2 wild-type pcDNA 3.1(B) myc/His (−) construct; Nr2f2^mutant: NRK52E cells transfected with Nr2f2 mutant pcDNA 3.1(B) myc/His (−) construct. (b) Cell extracts were subjected to immunoprecipitation (IP) with Nr2f2 antibody (Nr2f2 Ab1). The immune complexes were captured using agarose G beads. The immune-precipitated protein samples were denatured, resolved by SDS–polyacrylamide gel electrophoresis and transferred on to a polyvinylidene difluoride membrane. The membrane was probed by western blotting (WB) with anti-Fog2 antibody and anti-His-Tag antibody. A representative of four experiments is shown. Lanes labelled from the left—IgG: IgG antibody control; empty vector: NRK52E cells transfected with empty pcDNA 3.1(-B) myc/His vector; Nr2f2^wt: NRK52E cells transfected with Nr2f2 wild-type pcDNA 3.1(B) myc/His (−) construct; Nr2f2^mutant: NRK52E cells transfected with Nr2f2 mutant pcDNA 3.1(B) myc/His (−) construct; control: NRK52E cell lysate. Corroborative data obtained from Histidine-tagged Nr2f2 overexpressed in COS-7 cells are presented in Supplementary Fig. 3.