Figure 1.

CARP-1 binds with NEMO, and CARP-1 amino acids 553–599 and NEMO amino acids 221–261 harbor respective epitopes for interaction of CARP-1 and NEMO proteins. A, protein complexes from the indicated cells were immunoprecipitated with the noted antibodies followed by the analysis of the immunocomplexes by Western blotting (WB) using anti-NEMO (upper) antibodies. The membrane-containing proteins from whole-cell lysates were then probed with anti-CARP-1 (middle) or anti-NEMO (lower) antibodies for the presence of respective proteins. B, WB analysis of IP protein complexes was derived by using the indicated antibodies from the noted cell lines. The membrane-containing IP proteins were probed with anti-CARP-1 antibodies (upper), and the membrane-containing proteins from whole-cell lysates were probed with anti-NEMO (middle) or anti-CARP-1 (lower) antibodies for the presence of respective proteins. Arrowheads on the left or right, respectively, indicate the presence of the proteins and the molecular weight markers in A and B of each blot. Schematic of CARP-1 WT and its various mutants (C) and NEMO WT and its mutants (D) were utilized in co-IP–WB experiments to elucidate CARP-1 and NEMO interactions and to map the respective minimal epitopes. All CARP-1 proteins have Myc and His₆ epitopes at their C termini. All the NEMO proteins, with the exception of 2–260 and 221–261 mutants, harbored 6× Myc epitope at their N termini. NEMO 2–260 and 221–261 mutants had GST epitope at their N termini. Positive interactions are indicated by + and loss/absence of interaction is denoted by −.