Skip to main content
. 2009 Jul 28;14(6b):1555–1568. doi: 10.1111/j.1582-4934.2009.00866.x

Fig 8.

Fig 8

Ubiquitination of JAB1. (A) His-tagged ubiquitin was co-transfected with flag-JAB1 or flag-IKK2 as indicated followed by immunoprecipitation with anti-flag-beads and immunoblotting with anti-His- or anti-flag tag antibodies. A higher amount of ubiquitinated JAB1 or IKK2 was detected when the proteasome inhibitor MG132 was added. TNFα treatment (30 min, 50 ng/ml) resulted in increased ubiquitination. (B) ubiquitination of JAB1 was assessed after transfection with His-tagged ubiquitin and flag-tagged JAB1 in presence or absence of myc-tagged IKK2 and MG132. His-Ubiquitinated proteins were purified by NiNTA-resin under chaotropic conditions (including guanidine-HCl as described in the ‘Materials and methods’ section), preventing co-precipitation of non-covalently linked proteins. Bound His-ubiquitinated proteins were washed, followed by SDS-PAGE and Western blotting with anti-flag antibodies. (C) ubiquitination of JAB1 was analysed as in (A) with either wild-type IKK2 (flag-IKK2wt), kinase-deficient IKK2 mutant (flag-IKK2mut) or constitutive active IKK2 (HA-IKK2ca). Western blotting was carried out against the myc-tag of JAB1.