The formation of NEMO dimers requires Cys54 and Cys347. (A) NEMO is a 419 amino acid protein with 11 Cys residues, which are indicated. CC1, CC2, coiled coil domains; LZ, a leucine zipper; ZF, zinc finger. (B) Expression vectors for the indicated FLAG-NEMO proteins (wt, wild-type) were transfected into NEMO-deficient fibroblasts. Whole cell extracts were separated under non-reducing conditions and immunoblotted for NEMO. (C) Stable cell lines expressing NEMO or NEMO-C54/347A (see Fig. 3) were treated for 2 h with the small-molecule EqM. Whole cell extracts were prepared and immunoblotted for NEMO after conventional reducing SDS-polyacrylamide gel electrophoresis. NS, non-specific band.

Molecular modeling of the NEMO dimer predicts that Cys54 and Cys347 can form intermolecular disulfide bonds. Light blue and light purple ribbons represent each NEMO monomer. Red, disulfide-bonded cysteines; gold, residues involved in the leucine zipper motif (Leu 322, 329, 336, 343); N, N terminus; C, C terminus.

NEMO forms disulfide-bonded dimers. (A) NEMO-deficient fibroblasts were transfected with pcDNA-FLAG-NEMO. Total cell extracts were prepared in AT lysis buffer and samples were resuspended in SDS sample buffer with or without β-mercaptoethanol (β-Me), as indicated. Samples were heated for the indicated amount of time at 95°C. An anti-NEMO Western blot was then performed. FLAG-NEMO is the monomeric form of NEMO (~47 kDa) and FLAG-NEMO² migrates at approximately twice the molecular weight of FLAG-NEMO. (B) NEMO-deficient fibroblasts were transfected with expression plasmids for vector alone (pcDNA), FLAG-NEMO or Myc-NEMO as indicated. Cell extracts were heated at 95°C in SDS sample buffer with (left four lanes) or without (right four lanes) β-Me. Samples were analyzed by anti-NEMO Western blotting. (C) HeLa cell extracts were prepared in AT lysis buffer containing 20 mM N-ethylmaleimide (NEM) and with the indicated concentration of DTT (top). Samples were then boiled in SDS sample buffer (SB) with the indicated final concentrations of DTT. Samples were analyzed by anti-NEMO Western blotting. (D) Total cell extracts from the indicated cell lines were prepared in AT lysis buffer with 20 mM NEM. Samples were separated by reducing (left panel) or non-reducing (right panel) SDS polyacrylamide gel electrophoresis, and anti-NEMO Western blotting was then performed.

Comparison of wild-type and C54/347A NEMO. (A) Retroviral vectors containing no cDNA (pBABE), wild-type NEMO or C54/347A NEMO and the puromycin resistance gene were used to infect NEMO-deficient mouse fibroblasts. An anti-NEMO Western blot was then performed on stable pools of selected cells. Vector, pBABE. (B) Serum-starved stable cell lines expressing wild-type or C54/347A mutant NEMO were left untreated (0) or were treated with TNFα for the indicated amounts of time and an EMSA was performed using a κB-site probe (upper panel). In the lower panel is an anti-NEMO Western blot taken from the same extracts. (C) Stable cell lines expressing NEMO or NEMO-C54/347A were treated with the indicated concentrations of H₂O₂ for 10 min. Whole cell extracts were prepared (with 20 mM NEM) and separated by SDS-PAGE under non-reducing conditions. Samples were immunoblotted for NEMO. (D) Serum-starved NEMO-reconstituted cells were untreated or pretreated with H₂O₂ for 10 min and were then treated with TNFα for 7.5 min. An immune complex IKK kinase assay using GST-IκBα(1-55) was performed. Shown is ³²P-labeled GST-IκBα.