Fig. 5. Biochemical analysis of the Myc-Lin-2/Pals1 PDZ dominant-negative chimeric protein. (A) Schematic diagram illustrating the domain organization of wild-type Myc-Lin-2, Myc-Lin-2 missing the PDZ domain (Myc-Lin-2PDZ), and the Myc-Lin-2 in which the PDZ domain was replaced with the Pals1 PDZ domain (shown in black). Recognized domains are denoted above the protein schematics: CKII, calmodulin serine/threonine kinase II like domain; L27, Lin-2/Lin-7 domain; PDZ, PSD-95/Discs Large/ZO-1 domain; SH3, Src homology 3 domain; 4.1B, protein 4.1 binding domain; and GUK, guanylate kinase domain. (B) Full length Myc-Lin-2 and the Myc-Lin-2/Pals1 PDZ chimera constructs were individually expressed in HEK293 cells. Pulldown experiments with GST (negative control) and GST fused to the last 20 residues of CRB3 were performed on the lysates prepared from the transiently transfected 293 cells. (C) Lysates were prepared from MDCK stable cell lines expressing the indicated proteins. Lysate proteins and anti-Myc immunoprecipitates were resolved by SDS-PAGE and the proteins visualized via anti-Myc immunoblot. (D) Myc-Lin-2/Pals1 PDZ chimeric protein and Myc-Lin-2 missing the PDZ domain were immunoprecipitated from MDCK cells. Co-precipitated endogenous CRB3 was revealed by immunoblotting with anti-CRB3 antisera.

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