Figure 2.

Binding of CaM-KII to CaM-KIIN. (A) GST/CaM-KIIN precipitation of CaM-KII. Either recombinant CaM-KIV, CaM-KII, or soluble brain extract was incubated with GST/CaM-KIIN in the presence of Ca²⁺/CaM or EGTA, precipitated with glutathione-Sepharose, washed, and separated by SDS/PAGE. Samples then were visualized by Coomassie staining or by Western blot with anti-CaM-KII antibody. The positions of CaM-KII (upper arrow) and GST/CaM-KIIN (lower arrow) are indicated. The Western blot of the soluble brain extract precipitation (Right) visualized both the α and β subunits of CaM-KII. (B) Coimmunoprecipitation of CaM-KII and CaM-KIIN. Rat forebrain supernatant was split into 200-μl aliquots and incubated with either protein A Sepharose alone (first two lanes) or with a mAb CaM-KIIα in the presence of 3 mM CaCl₂ or 1 mM EGTA. After centrifugation the immune complex was separated by SDS/PAGE and immunoblotted with the CaM-KIIN antibody (Upper Left) or with preadsorption with the antigenic peptide (Upper Right). The membrane then was immunoblotted with the polyclonal CaM-KII Ab showing both the α and β isoforms (Lower). (C) Coimmunoprecipitation of CaM-KIIN with CaM-KII from transfected cells. HEK 293 cells were transiently transfected with CaM-KIIN, CaM-KII (His-282–Arg), or together and subsequently lysed. CaM-KII was immunoprecipitated from the soluble cell extract with 3 mM Ca²⁺ and separated by SDS/PAGE. The samples then were immunoblotted with anti-CaM-KIIN. (D) Colocalization of active CaM-KII and CaM-KIIN with transfected cells. COS-7 cells were transiently transfected with the indicated combinations of GFP, GFP/CaM-KIIN fusion protein (GFP-KIIN), wild-type CaM-KII, or activated CaM-KII (H282R). Cells were visualized for GFP (Upper) or for activated CaM-KII by using a Thr²⁸⁶ phosphospecific-antibody (Lower).