Figure 2. Characterisation of the ubiquitylation status and protein levels of CYLD and MIB2.

(A) MIB2 ubiquitylates CYLD, resulting in its degradation and a reduction in levels of Notch ligand JAG2. HA-MIB2, CYLD (untagged) and His₆-ubiquitin constructs were co transfected in HEK-293T cells and drove the ubiquitylation of CYLD compared to cells transfected with CYLD/His₆-ubiquitin alone (panel (i), compare lanes 1 and 2). Co-transfection of CYLD/HA-MIB2-ΔRING/His₆-ubiquitin prevented auto-ubiquitylation of MIB2 (panel (iii), compare lanes 2 and 3) and did not result in ubiquitylation of CYLD (panel (i), compare lanes 2 and 3). Overexpression of MIB2 but not MIB2-ΔRING appeared to decrease endogenous levels of its downstream target, JAG2 (panel (iv)). Tubulin was used as a loading control (panel (v)). (B) CYLD expression is associated with increased ubiquitylated MIB2 protein and reduced JAG2 protein, independent of its ubiquitin hydrolase activity. Lysates from transfected cells prepared in denaturing buffer were analysed by western blot and purified by nickel chromatography and showed that in the absence of exogenous CYLD expression, overexpression of HA-MIB2/His₆-ubiquitin predominantly resulted in high order molecular weight (MW) products at around 150 kDa (panel (i), lane 2) and decreased MIB2 in the whole cell lysate (panel (ii), lane 2 compared to lane 4 and 5). In the presence of exogenous CYLD expression, a larger proportion of lower MW ubiquitylated MIB2 products were observed (panel (i) compare lane 2 vs. lane 4). In order to determine whether this was dependent on CYLD deubiquitylase activity, HA-MIB2/His₆-ubiquitin was co-expressed with a catalytically inactive CYLD (CYLD-C601A, with no apparent difference in poly-ubiquitylated MIB2 levels versus co-expression with wild type CYLD seen (panel (i), compare lane 4, 5 and MIB2 levels in whole cell lysate, panel (ii), lanes 2, 4 and 5). Tubulin was used as a loading control (panel (v)).