Figure 5: The association of Bach2 with Batf family proteins.
From: Bach2–Batf interactions control Th2-type immune response by regulating the IL-4 amplification loop
(a) Immunoprecipitation (i.p.) and immunoblot (i.b.) analyses of the association of Bach2 and Batf in 293 T cell lysates left untransfected (−) or transfected (+) to express Myc-tagged Batf (Myc-Batf) and/or HA-tagged Bach2 (HA-Bach2; left) or to express Myc-tagged Batf (Myc-Batf3) and/or HA-tagged Bach2 (HA-Bach2; right); below (Input), a parallel analysis of the total cell lysates (without i.p.). The data are representative of three-independent experiments with similar results. (b) A pull-down assay of the binding of the Batf/Bach2 complex to an AP-1 consensus oligonucleotide in 293 T cell lysates transfected to express Myc-Batf and/or HA-Bach2 (Input (below), i.b. analysis of whole-cell lysates without precipitation). Wedges indicate 3-fold ‘titration’ of the input lysates. The data are representative of independent experiments with similar results. (c) The results of the pull-down assay of the binding of Bach2–Batf complex to wild-type (WT) or mutant (Mut.) AP-1 consensus oligonucleotides in the effector CD4 T cell lysates. The data are representative of three-independent experiments with similar results. (d) A schematic representation of the Bach2 mutants (left). The results of the AlphaScreen to detect the interaction of Batf with the Bach2 mutants (right). The relative intensities to dihydrofolate reductase (DHFR) binding are presented as the averages of three-independent experiments. (e) i.p. and i.b. analyses of the association of the wild-type Batf with Bach2 point mutants (L687A and L694A) (left) or wild-type Bach2 with Batf point mutants (L68A and L61A) (right l) in 293 T cell lysates. The cell lysates were prepared as described in (a). The data are representative of three-independent experiments with similar results.
