PDI co-precipitates with TDP-43 and is increased in mutant TDP-43 transgenic mouse spinal cords.

(A) Co-immunoprecipitation of wildtype and mutant Q331K TDP-43-mCherry from Neuro2a cell lysates using an anti-PDI antibody followed by TDP-43 immunoblot. Control immunoprecipitations were performed using untransfected (Untrans) or mCherry expressing cell lysates, buffer only with coprecipitating antibodies or TDP-43 Q331K cell lysates using an irrelevant, isotype-matched control antibody (IgG). Input control (2%) shows expression of mCherry-TDP43 in wildtype and Q331K cell lysates. (B) Co-immunoprecipitation of PDI-V5 from wildype and mutant A315T and Q331K TDP-43-mCherry from HEK293T cell lysates using an anti-RFP antibody to precipitate mCherry followed by TDP-43 and V5 immunoblot. Control immunoprecipitations were performed using Untrans cell lysates or mCherry and PDI-V5 expressing cell lysates, buffer only with coprecipitating antibody or Q331K TDP-43 and PDI-V5 co-transfected cell lysates using an irrelevant, isotype-matched control antibody (IgG). Input control shows expression of PDI-V5, TDP-43-mCherry and endogenous (End.) TDP-43. Approximate molecular weight markers are shown on the right, and representative images are shown. (C) Proteins were extracted from spinal cords of three mutant A315T TDP-43 transgenic mice and three litter-matched non-transgenic controls, and immunoblotting was performed for PDI, FLAG-TDP-43 (detected using antibody against FLAG), and β-actin. (D) Quantification of PDI levels from immunoblots normalised to β-actin by densitometry. Data represent mean normalised values from three independent analyses and are shown as mean ± SEM. *p<0.05 versus non-transgenic controls by unpaired two-tailed t-test. (E) Immunohistochemistry for TDP-43 and PDI in non-transgenic and A315T mutant TDP-43 mouse spinal cords. Cells were immunostained for TDP-43 (first column, green), PDI (second column, red), and were stained using To-Pro to identify nuclei (third column, blue). Merged images (fourth column) are also shown. Increased co-localisation of PDI with TDP-43 is seen in A315T animals compared to non-transgenic controls (Non-trans). All scale bars represent 10 µm. (F) Quantification of the percentage of motor neurons in which PDI and TDP-43 were co-localised. A total of 60 cells per group were counted, and data represent mean values from two mice per group.