y upstream modulator of ER stress. We also demonstrate that C-terminal TDP-43 fragments form inclusions in close association with PDI and the ER/Golgi apparatus, suggesting that TDP-43 inclusion formation causes dysfunction of ER/Golgi components. Moreover, over-expression of mutant TDP-43, and to a lesser extent wildtype TDP-43, induced ER stress via several UPR pathways, including activation of XBP-1 and ATF6, thus linking ER stress to neurodegeneration. Finally we show up-regulation of PDI in the spinal cords of transgenic mutant A315T TDP-43 mice, and interaction of mutant TDP-43 with PDI, providing further evidence of an ER-associated protective response in TDP-43 proteinopathies. Materials and Methods DNA constructs Site-directed purchase SB366791 mutagenesis was performed to remove the stop codon and insert a unique BamHI restriction enzyme site into a pcDNA3.1 Myc-tagged human TDP-43-encoding construct. The Myc-TDP-43 sequence was inserted between HindIII and BamHI restriction sites in pmCherry.N1, to allow expression of human TDP-43 with a C-terminal mCherry tag. The subsequent 22576162 wildtype Myc-TDP-43-mCherry construct was used as a template to produce six ALS-linked mutant TDP-43 expressing vectors by QuikChange site-directed mutagenesis according to 7884917 the manufacturer’s instructions. The 218414 TDP43-mCherry construct was produced by cloning of a PCR product from a primer incorporating a unique HindIII restriction enzyme site immediately 59 to the M218 codon of TDP-43 into a pCR2.1TOPO vector with subsequent sub-cloning into pmCherry.N1 using HindIII and BamHI restriction sites. EGFPTDP-43 constructs were as described previously. For the detection of ER stress, an ATF6-EGFP reporter construct was used. For co-immunoprecipitation experiments, a vector encoding PDI with a V5 epitope tag was used. Cell culture Mouse neuroblastoma Neuro2a, human epithelial HeLa and human embryonic kidney HEK293T cell lines were maintained in high glucose DMEM with 10% heat-inactivated fetal calf serum, 100 mg/mL penicillin and 100 mg/mL streptomycin. Cells were treated as indicated with thapsigargin to induce ER stress, MG132 to inhibit proteasomal function, cycloheximide as a general translation inhibitor, and arsenite as a standard oxidative stress inducer of SGs. For immunocytochemistry, cells were plated on poly-L-lysine-coated 12 mm glass coverslips. Cells were transfected with constructs using Lipofectamine2000 with PLUS reagent according to the manufacturer’s protocol. Cell lysates were collected in TN buffer with 0.1% SDS, 1% protease inhibitor cocktail and 1% phosphatase inhibitor by incubation on ice for 10 min. The supernatant was cleared by centrifugation at 16,100 g for 10 min. Protein concentrations of cell and tissue lysates were determined using the BCA protein assay by comparison with bovine serum albumin standards. Immunocytochemistry and microscopy Cells were fixed with 4% paraformaldehyde at room temperature for 15 min, permeabilised with 0.1% triton X-100 in PBS for TDP-43 Induces ER Stress 10 min and then blocked with blocking buffer for 30 min at room temperature. Primary antibodies were diluted in blocking buffer and incubated overnight at 4uC. Primary antibodies were: mouse anti-PDI 
Ab2792, rabbit anti-TDP-43 10782, mouse anti-HuR 39-0600, rabbit anti-ERGIC53 E1031, rabbit anti-XBP-1 sc-7160, mouse antiGADD153/CHOP sc-7351 and mouse anti-GM130 610823. Cells were washed in PBS and then incubated with secondary antibody at 1:5000 in PBS for 1 h at
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