Pelleted cells were lysed in RIPA buffer (50 mM Tris pH 7.4; 150 mM NaCl; 1 mM EDTA; 1% NP-40; 0.1% SDS; 0.1% sodium deoxycholate; 1 mM PMSF; PhosSTOP and cOmplete PIC (Roche)) sonicated, and quantified by BCA assay. Equal sample amounts were then immunoblotted using Bolt gels and buffers (Thermo Fisher). Blots were blocked in 5% non-fat dry milk in TBSt (0.05% tween), washed in TBSt and incubated overnight at 4 °C with the following antibodies: anti-TDP-43 (ProteinTech; 12,892–1-AP; 10,782–2-AP); anti-Actin (Millipore; MAB1501); anti-α-synuclein (BD 610787); anti-ATXN2 (BD Biosciences; 611,378); anti-VCP (Thermo.; MA3–004); anti-AHR (Thermo.; MA1–514); anti-α-tubulin (Sigma-Aldrich; T5168). After washing, HRP-conjugated secondary antibodies (Jackson) were incubated with the blots the following day. Blots were activated with Pierce ECL chemiluminescent substrates (Thermo Fisher) and imaged using a ChemiDoc XRS+ Imager (BioRad). Band densitometries were assessed using Image Lab Software (BioRad).
RNA was collected from cultured cells by RNeasy minikit (Qiagen). cDNA was generated using High-Capacity cDNA Reverse Transcriptase (ABI). qPCR was performed using iQ SYBR green Supermix (Bio-Rad) on a 7900HT Fast Real-Time PCR system and the data was analyzed on SDS software. qPCR primer sequences are available in Additional file1 : Table S1.
RNA was collected from cultured cells by RNeasy minikit (Qiagen). cDNA was generated using High-Capacity cDNA Reverse Transcriptase (ABI). qPCR was performed using iQ SYBR green Supermix (Bio-Rad) on a 7900HT Fast Real-Time PCR system and the data was analyzed on SDS software. qPCR primer sequences are available in Additional file