Rat anti tdp 43 2h4

N2a cells have been transfected with pFN33_TDP-43 and pFN35_TDP-43 in 10 cm dishes either treated with 0.1% DMSO as a vehicle control or 10 µM auranofin for 60 min at 37 °C before collecting the cells. Cells were extracted with PBS + protease and phosphatase inhibitors (cOmplete^TM Protease Inhibitor Cocktail and PhosSTOP, Roche, Basel, Switzerland) assisted by ultasonication. Supernatant (200 µl) of a 10 min 10,000 xg centrifugation step was applied to a Superose 6 10/300 GL column and eluted at 0.2 ml/min on an ÄKTApurifier (GE Healthcare Life Sciences, Freiburg, Germany) collecting 500 µl fractions. 50 µl of each fraction supplemented with 50 µl 0.1 M carbonate buffer pH 9.6 were used to coat 96-well Nunc MaxiSorp microplates overnight. After washing and blocking with 0.5% cold fish gelatine, wells were probed with rat anti-TDP-43 2H4 (BioLegend, San Diego, CA, USA). In addition, we used anti-TIAR1 (#8509, New England Biolabs, Ipswich, MA, USA) as a stress granule protein marker. The detection was performed in single well per fraction with HRP-conjugated rabbit anti-rat antibody (1:10000, DAKO GmbH, Jena, Germany) and tetramethylbenzidine (AJ Roboscreen, Leipzig, Germany).

N2a cells have been transfected with pFN33_TDP-43 and pFN35_TDP-43 construct as described above and incubated for 24 h at 37 °C. Cells have been lysed with 500 µl Laemmli lysis buffer per 2 wells of a 12-well plate (Tris/HCl, pH 6.8, 62.5 mM, 10% glycerol, 2% SDS, 5% β-mercaptoethanol, 0.1 M DTT). Proteins were separated using a 12% SDS-PAGE and subsequently transferred to a polyvinylidene difluoride (PVDF) membrane (PolyScreen; PerkinElmer Life Sciences, Boston, MA). Membranes were washed once in TBS-T buffer (150 mM NaCl, 20 mM Tris/HCl pH 7.4, 0.05% Tween 20), blocked in TBS-T containing 1% BSA (w/v), and probed with rat anti-TDP-43 2H4 (1:2000, BioLegend, San Diego, CA, USA). Detection of bound primary antibody was performed with rabbit anti-rat antibody (1:10000, DAKO GmbH, Jena, Germany). Blots were developed with ECL reagents and imaged in ECL-Imager (DNR MF-ChemiBIS Image Analysis System 1.6, DNR Bio-Imaging Systems, Biostep, Germany).