Fig. 1.

NF-κB activation induced by hUBQLN2 is suppressed with hUBQLN2 siRNA. Neuro2A cells were transfected with control plasmid, pCMV-hUBQLN2^WT or pCMV-hUBQLN2^P497H. Cells were treated or not with TNF-α 20 ng/ml for 4 h before luciferase assay or before collecting the cells. a Cytoplasmic (C) and nuclear (N) extraction was realized 48 h after transfection. Antibodies were use according to Materials and Methods. b Immunoblot quantification of nuclear phospho-NF-κB p65 vs p84 nuclear matrix as compared to nuclear levels in control cells (p = 0.0426, n = 3). c Neuro2A cells were stably transfected with pluc2p-NFκB-RE plasmid and then transfected with control plasmid, pCMV-hUBQLN2^WT or pCMV-hUBQLN2^P497H. Luciferase activity (NF-κB) was measured at 24 h (s < 0.0001 for hUBQLN2^WT and hUBQLN2^P497H, n = 6,) and (d) at 48 h (p = 0.0004 for hUBQLN2^WT and p = 0.0061 for hUBQLN2^P497H, n = 3). e Cells were co-transfected with control plasmid, pCMV-hUBQLN2^WT or pCMV-hUBQLN2^P497H and with hUBQLN2 siRNA or scrambled siRNA. Luciferase activity was measured at 48 h after transfection (p < 0.01, n = 5). f Immunoblot of UBQLN2 proteins. Upper band corresponds to mouse UBQLN2 (endogenous) and lower band to human UBQLN2 (transfected). Immunoblot was showing the reduced levels of only human UBQLN2 after co-transfection with siRNA