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Fig. 3 | Molecular Brain

Fig. 3

From: Ubiquilin-2 drives NF-κB activity and cytosolic TDP-43 aggregation in neuronal cells

Fig. 3

hUBQLN2 co-localized with TDP-43 and P62 but not with NF-κB and IκB-α. Immunofluorescence of Neuro2A cells at 48 h after transfection with (a) control plasmid, (b) pCMV-hUBQLN2WT and (c) pCMV-hUBQLN2P497H. Indicated antibodies were used according to Materials and Methods. Microscopy pictures were taken at 63× magnification. d-e Western analysis from three repeated experiments was used for quantification of (d) TDP-43 level (p = 0.0227 for hUBQLN2WT and p = 0.0032 for hUBQLN2P497H) and (e) p62 level (p = 0.0058 for hUBQLN2WT and p = 0.0130 for hUBQLN2P497H) in insoluble fraction. Results were calculated in fold change compared to proteins level in insoluble fraction of control cells. f Neuro2A cells were transfected with control plasmid, pCMV-hUBQLN2WT or pCMV-hUBQLN2P497H. Cells were extracted 48 h after transfection using aggregates assay protocol. The results suggested that overexpression of UBQLN2 species increased levels of UBQLN2, TDP-43 and p62 in the insoluble fraction. g Live imaging was performed on Neuro2A pFeGFP-hUBQLN2P497H and DsRED-TDP-43 co-transfected cells. Microscopy pictures were taken at 24 and 48 h after transfection. h Immunoblot of TDP-43 in cytoplasmic (C) vs nuclear (N) after transfection with control plasmid, pCMV-hUBQLN2WT or pCMV-hUBQLN2P497H plasmids. i HEK293 cells and Neuro2A cells were transfected with either control plasmid or pCMV-hUBQLN2P497H plasmid. Protein levels of hUBQLN2 and mUBQLN2 were measured at 48 h after transfection. Human UBQLN2 levels were similar in control HEK293 and pCMV-hUBQLN2P497H Neuro2A transfected cells. Scale bar = 25 μm

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