Figure 8: Co-immunoprecipitation of barr2/CAMKII complexes.
(a) Studies with wt mouse pancreatic islets. Lysates prepared from wt mouse pancreatic islets were subjected to immunoprecipitation with either an anti-CaMKIIδ antibody or goat IgG (negative control). Immunoprecipitated proteins were then probed with anti-barr2, anti-CaMKII (pan), and anti-CaV1.2 (α1-subunit69) antibodies. Note that barr-2, but not CaV1.2, could be co-immunoprecipitated with CAMKII. (b) Co-immunoprecipitation studies with transfected HEK293T cells. HEK293T cells were transfected with the indicated plasmids. All barr2 constructs carried an N-terminal HA epitope tag (HA-barr2-N-term: barr2 residues 1–181; HA-barr2-C-term: barr2 residues 180–408). In addition, cells were infected with an adenovirus coding for flag-CaMKIIδ (lanes 2–5) or an empty control virus (lane 1). Flag-CaMKIIδ was immunoprecipitated from cell lysates with an anti-flag antibody, followed by immunoblotting studies to detect co-immunoprecipitated barr2/barr2 fragments. This analysis indicated that the barr2-N-domain (similar to full-length barr2), but not the barr2-C-domain, was clearly detectable in the immunoprecipitates. The blots shown are representative of two or three independent experiments.
