Fig. 6.

M1-Ub chain formation and activation of the canonical IKK complex in cells from knock-in mice expressing an E3 ligase-inactive mutant of HOIP. (A) MEFs expressing either wild-type (WT) HOIP or the E3 ligase-inactive HOIP[C879S] mutant ([C879S]) were stimulated with either 5 ng/ml IL-1α or 10 ng/ml mTNFα for the times indicated. M1-Ub chains captured from the cell extracts with Halo-NEMO were identified by immunoblotting with a specific antibody. (B, C) Macrophages derived from the foetal livers of WT or HOIP[C879S] knock-in mice were stimulated with poly(I:C) (B) or LPS (C) for the times indicated. Cell extract (20 μg protein) was subjected to SDS-PAGE and immunoblotted with antibodies that recognize M1-Ub chains (top panel), the activated forms of IKKα, IKKβ, JNK1 and JNK2, the three components of LUBAC (HOIP, HOIL-1 and Sharpin) and GAPDH as a loading control. (D) As in A except that the extracts (20 μg protein) were immunoblotted with antibodies that recognize the active phosphorylated form of IKKα and IKKβ and for GAPDH as a loading control.