Figure 2

Anti-C2Cat antibodies preferentially recognize active cPKCs.

(A) Reactivity of sera obtained from rabbits immunized with C2Cat-KLH to C2Cat-peptide coupled to BSA was determined by ELISA as described in Methods (n = 3). (B) SDS PAGE and Coomassie blue staining of lysates from induced bacteria expressing cPKC C2-GST (lane1), purified C2-GST (lane 2), induced bacteria expressing GST (lane 3) and purified GST (lane 4). (C) Recognition of cPKC C2-GST domain and GST alone by anti-C2Cat was evaluated by ELISA assay. Anti-C2Cat was diluted 1:50 (n = 3). (D) ELISA assay used to detect the reactivity of anti-C2Cat to active cPKC isoenzymes α, βΙ, βΙΙ and γ (with phospholipids and calcium)/inactive cPKC (100 ng) compared to commercially available anti-PKCs: α, βΙ, βΙΙ, γ antibodies that recognizes the isoenzyme specific V5 domains (1:500) (n = 3). (E) ELISA assay used to detect the reactivity of anti-C2Cat to cPKCs (α, βΙ, βΙΙ and γ) and nPKCs (δ and ε) (n = 3). Statistics was determined using ANOVA-Bonferroni test where ***p < 0.001 and **p < 0.01.