Binding to Lys63-linked or linear polyubiquitin chains is disrupted by a point mutation in the UBAN of ABIN1 or NEMO. (A and B) The binding of WT and mutant human ABIN1 and NEMO to polyubiquitin chains is described in Materials and methods. Polyubiquitin chains captured by the immobilized proteins were released by denaturation in 1% (wt/vol) SDS, subjected to SDS-PAGE and immunoblotted with an anti-ubiquitin antibody (Dako). (A) Lanes 1 and 6 show, respectively, the K48-linked and K63-linked polyubiquitin preparations used in the experiment. The binding of K48-linked (lanes 2 and 3) and K63-linked (lanes 7 and 8) polyubiquitin chains to WT ABIN1 (left) or NEMO (right) and to the ABIN1[D472N] and NEMO[D311N] mutants (lanes 4, 5, 9, and 10, left and right) is shown. Similar results were obtained in two different experiments. (B) As in A, except that binding to linear polyubiquitin oligomers was studied. Lanes 1–3 show the di-ubiquitin and nona-ubiquitin preparations used in the experiment. Lane 4 shows the lack of binding to di-ubiquitin, lane 6 the binding to nona-ubiquitin, and lane 8 the selective capture of nona-ubiquitin by ABIN1 and NEMO when presented with a mixture of nona-ubiquitin and di-ubiquitin, and lanes 5, 7, and 9 the lack of binding of ABIN1[D472N] and NEMO[D311N] to linear ubiquitin oligomers. Similar results were obtained in two different experiments. Gray lines indicate that intervening lanes were spliced out. (C) BMDM from ABIN1[D485N] and WT mice were stimulated with 100 ng/ml LPS for the times indicated. ABIN1 was immunoprecipitated from the cell extracts and immunoblotted with anti-IRAK1 (top), anti-ubiquitin (middle), and anti-ABIN1 (bottom) antibodies. Similar results were obtained in two different experiments.