Journal: Molecular Brain

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

doi: 10.1186/s13041-015-0162-6

Figure Lengend Snippet: 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-hUBQLN2 WT and ( c ) pCMV-hUBQLN2 P497H . 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 hUBQLN2 WT and p = 0.0032 for hUBQLN2 P497H ) and ( e ) p62 level ( p = 0.0058 for hUBQLN2 WT and p = 0.0130 for hUBQLN2 P497H ) 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-hUBQLN2 WT or pCMV-hUBQLN2 P497H . 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-hUBQLN2 P497H 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-hUBQLN2 WT or pCMV-hUBQLN2 P497H plasmids. i HEK293 cells and Neuro2A cells were transfected with either control plasmid or pCMV-hUBQLN2 P497H plasmid. Protein levels of hUBQLN2 and mUBQLN2 were measured at 48 h after transfection. Human UBQLN2 levels were similar in control HEK293 and pCMV-hUBQLN2 P497H Neuro2A transfected cells. Scale bar = 25 μm

Article Snippet: Primary antibodies were monoclonal Flag M2 (Sigma-Aldrich, Saint-Louis, 1:100), TARDBP (Proteintech, Chicago, 1:600), NF-κB p65 (Santa-Cruz, 1:200), IκB-α (Santa-Cruz, 1:200) and SQSTM1/P62 (Cell signaling, 1:200).

Techniques: Immunofluorescence, Transfection, Control, Plasmid Preparation, Microscopy, Western Blot, Over Expression, Imaging