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Cerebella were dissected from mice and lysed in RIPA lysis buffer [50mM Tris HCl, pH7.4, 150mM NaCl, 1% sodium deoxycholate, 1% NP-40, 0.2% SDS, phosphatase (Sigma) and protease inhibitors cocktail (Roche)]. After three cycles of freeze and thaw, proteins were separated on a 12% or 15% SDS-PAGE gel and transferred onto a nitrocellulose membrane. The following primary antibodies were used: anti-ATXN1 (rabbit 11NQ, Orr lab), anti-PSD95 (Biolegend), and alpha-tubulin (mouse, Sigma). Signals from secondary antibodies linked to horseradish peroxidase (HRP) (GE Healthcare) were detected using Amersham ECL Western Blotting Detection Reagent (GE Healthcare) and ImageQuant LAS 4000 imager (GE Healthcare); protein levels were quantified using ImageQuant (GE healthcare) and ImageJ. Data was analyzed with one-way ANOVA followed by Bonferroni post-hoc test.

Statistical evaluation of behavioral analysis, immunohistochemistry, and Western blotting results was performed using Prism 8 (GraphPad Software). For Rotarod test and body weight data, a two-way ANOVA with Fisher’s LSD test was used at each age. The Kaplan–Meier method was used to analyze survival and onset in each SOD1^G93A mouse strain. Immunohistochemistry and Western blotting results were analyzed using either ImageJ or ImageQuant software (GE Healthcare).

The Western blot analysis was performed as previously described [42 (link)]. In brief, cells were lysed with lysis buffer (50 mM Tris; pH 7.6; 150 mM NaCl; 5 mM EDTA; 1 mM Na₃VO₄; 10 mM NaF; 10 mM sodium pyrophosphate; 1% NP-40) containing protease inhibitor cocktail (0.01 M EDTA; 1 mM DTT; 1 mM Leupeptin; 1 mM PMSF; and 1 µM Aprotinin). The cell lysate was placed on ice for 60 min and centrifuged at 17,982× g for 15 min. The total protein was quantified using the Bradford method (Quick Start ^TM Bradford Protein Assay; Bio-Rad, Hercules, CA, USA). Total proteins were separated by 12% polyacrylamide gel and transferred onto Trans-Blot Turbo Midi Nitrocellulose Transfer Packs (Bio-Rad, Hercules, CA, USA). The membrane was incubated with primary followed by secondary antibodies after blocking with 5% non-fat milk. Immunodetection was performed using the ECL KIT (Amersham Biosciences, Uppsala, Uppland, Sweden) in ImageQuant LAS 4000 mini (GE Healthcare Life Sciences, Pittsburgh, PA, USA) and quantified by the platform for scientific image analysis ImageJ (NIH) [46 (link)]. The following antibodies were used: polyclonal anti-MTAP antibody (Proteintech, Rosemont, IL, USA) diluted 1:800 and anti-β actin antibody (Cell Signaling Technology, Danvers, MA, USA) diluted at 1:2000.

All DNA substrates (Figure 5—source data 1) were synthesized by Eurofins/MWG (Ebersberg, Germany), resuspended in annealing buffer (20 mM Tris-HCl pH 8.0, 50 mM NaCl, 0.1 mM EDTA), annealed by heating to 85˚C for 5 min and slow-cooling to room temperature. Different amounts of GEN1 proteins (as indicated) were mixed with 40 nM 6FAM-labeled DNA substrates in 20 mM Tris-HCl pH 8.0, 50 ng/μl bovine serum albumin (BSA) and 1 mM DTT. Reactions were initiated by adding 5 mM MgCl₂, incubated at 37°C for 15 min and terminated by adding 15 mM EDTA, 0.3% SDS and further, DNA substrates were deproteinized using 1 mg/ml proteinase K at 37°C for 15 min. Products were separated by 8% 1x TBE native polyacrylamide gel electrophoresis, the fluorescence signal detected with a Typhoon FLA 7000 phosphoimager (GE Healthcare), quantified with IMAGEJ (GE Healthcare) and visualized by GNUPLOT (Williams et al., 2015 ).

Immunoblot signals were quantified using Image J or ImageQuant (GE Healthcare) software, for quantification of cell surface PrP levels by FACS analysis mean fluorescence values were used for statistical evaluation. All values were expressed as percentage of the control value (100%). For comparison of multiple groups, one-way ANOVA followed by post hoc analysis with Dunnett’s multiple comparison test was used. For pairwise comparisons, nonparametric Mann-Whitney tests were used. Statistical analysis was done using GraphPad Prism software.

For the PD assay, the different phospho-mimetic variants of cytoplasmic tails of human CD3ε were cloned into the pRP261 vector and fused C-terminally to GST. The E.coli strain BL21 was transformed with the corresponding vectors. After 3 h induction with 1 mM IPTG, bacteria were lysed. GST-CD3ε fusion proteins were purified using glutathione sepharose beads (GE Healthcare). CD3ε PD assay was performed overnight at 4°C. After washing, proteins were separated by SDS-PAGE and immunoblotted in a semi-dry chamber for 1 h at 18 V. Chemiluminescence was used to image protein bands using a CCD camera (ImageQuant LAS 4000; GE Healthcare). ImageJ and ImageQuantTL software were used to determine the relative band intensity (GE Healthcare).