Characterization of CKLiK antibody. (A) An antibody against the C-terminal portion of CKLiK was generated by immunizing rabbits with a Keyhole Limpet Hemocyanin (KLH)-coupled peptide as indicated. (B) Western blots with CKLiK antisera were performed. 32D cells (10⁶) stably expressing an N-terminal epitope-tagged CKLiK (32D HA-CKLiK) or control cells (32D) were lysed and separated by SDS-PAGE, and Western blotting was performed as described in “Materials and methods.” (C) Immune precipitations were performed with CKLiK antibody. 32D cells (10⁷) were lysed, and immune precipitations were performed with 1 μL and 0.2 μL purified CKLiK antibody (α-CKLiK) as indicated. CKLiK, as well as the immunoglobulin heavy chain (IgH) and immunoglobulin light chain (IgL), are indicated. (D) Immune-complex kinase assays were performed with or without Ca²⁺ and calmodulin, using the CKLiK antibody and an antibody against the epitope-tag (α-HA) as a control. CREMβ was used as a substrate.