Yeast strains and plasmids.
All yeast strains and plasmids used in this study are listed in Supplemental Tables 1 and 2, respectively. Methods to generate yeast strains and plasmids are reported in the Supplemental methods section. Primers used are listed in Supplemental Table 3–6.
Yeast media, transformation and yeast cell viability assays.
Procedures for yeast growth and manipulation were performed according to standard protocols [41, 42].
Yeast cells were grown in standard rich media (10 g/L Bacto-yeast extract, 20 g/L Bacto-peptone) supplemented with 20 g/L of glucose or galactose as carbon source or in synthetic minimal media (1.7 g/L yeast nitrogen base without amino acids, 5 g/L ammonium sulfate) containing 20 g/L of glucose, raffinose or galactose and lacking the specific nutrients allowing for selection of transformed yeast clones. Media components were from Difco (Thermo Fischer Scientific), and auxotrophic requirements were from Sigma-Aldrich. Induction of gene expression under Gal1 promoter control was obtained by growing transformed yeast cells in raffinose containing medium for 16 hours, followed by dilution of the cultures to an optical density at 600 nm light adsorption (OD600) of 0.1 and growth in galactose containing medium. Cells were routinely incubated at 30 °C.
The PEG/lithium acetate method was used to transform yeast  and then cells were plated on selective solid media.
Yeast cell viability was performed by measuring the ability of yeast cultures to grow on solid (spotting assays) and liquid media (OD600 culture turbidity measurements) . In particular, for spotting assays, yeast cells were grown overnight at 30 °C in liquid media (SD or YP) containing raffinose. Cultures were then normalized to OD600 = 1, serially diluted and spotted onto synthetic solid media containing glucose or galactose and the auxotrophic requirements as needed. Plates were maintained at 30 °C for 2–3 days. Conversely, OD600 measurements were performed in a Microplate Reader spectrophotometer (TECAN) at a 600 nm wavelength (bandwidth 9 nm) with 15 flashes and 200 µl of culture per well. Briefly, three yeast clones were grown overnight in a raffinose containing medium (non-inducing condition), diluted in triplicate to OD600 = 0.1 in a galactose containing medium (inducing condition), and grown for 24 h. OD600 was then recorded immediately after the transfer (t0) or after 24 h of growth (t24h), in the galactose medium, and the OD600 ratio (t24/t0) was calculated. Data were then normalized to the mean ratio of control cultures CEN.PX IMX672 (hereafter abbreviated as CENPK).
Microscopy analyses of S. cerevisiae cells.
For fluorescence microscopy observations, cells grown for 6 h in a galactose-containing liquid medium were used, while for cell shape analysis by brightfield microscopy cells were cultured on galactose containing solid substrate for 2 days. In both cases, before microscopy analysis, harvested cells were washed and resuspended in PBS, and then mounted on a coverslip with a thin agar slab as described previously . Cells were imaged using an inverted microscope (CTR6000, Leica) equipped with a Xenon lamp, a suited fluorescence excitation/dichroic/emission filter setting (λex = 488 nm for the excitation of GFP) and a computer-assisted charge-coupled device camera (Hamamatsu Orca flash 4.0), which allowed the acquisition of digital micrographs for either fluorescence or brightfield microscopy.
For morphometric measurements, the cell length-to-width ratio was calculated on digitalized images using the Fiji ImageJ software.
Whole protein extraction from S. cerevisiae cells.
The extraction of proteins from yeast cells for Western blot (WB) and mass spectrometry (MS) analyses was performed as described elsewhere  with some modifications. Briefly, yeast cells were harvested by centrifugation (6,000 x g, 10 min, 4 °C). Cells were then resuspended in 0.1 M NaOH and incubated at room temperature for 20 min. After pelleting, cells were finally lysed by vortexing (2 min) in a buffer containing 62.5 mM Tris-HCl (pH 6.8), 2.3% (w/v) SDS and 10% (w/v) glycerol (buffer O). Lysates were boiled (3 min) and, after centrifugation to remove cell debris, protein quantification was performed in the supernatant using a bicinchoninic acid (BCA) assay kit (Thermo Fisher Scientific), according to the manufacturer’s instructions.
Yeast sample preparation for tandem mass tag-based quantitative proteomics.
For tandem mass tag (TMT)-based quantitative proteomic analysis, protein extracts (50 µg) from three biological replicates of parental CENPK and TDP 1C yeast strains were processed according to the filter-aided sample preparation method , using filters with 10,000 Da as molecular weight cut-off (Sartorius). Briefly, filters were washed four times with 8 M urea and 100 mM triethylammonium bicarbonate (TEAB) buffer (Sigma-Aldrich) and then treated in 100 mM TEAB containing 25 mM dithiothreitol (45 min, 55 °C) to reduce disulphide bonds, and then in the same buffer (100 mM TEAB) containing 55 mM iodoacetamide (45 min in the dark, RT) to block reduced cysteine residues. Finally, filters were washed twice using 100 mM TEAB and, afterwards, protein digestion was performed by adding 100 µL of sequencing grade modified trypsin (20 µg/mL− 1 in 100 mM TEAB, pH 8.0) to each filter (18 h, 37 °C). After digestion, filters were subjected to two subsequent centrifugation step (14,000 × g, 10 min) adding 50 µL of 100 mM TEAB for peptide recovery. Peptide mixtures were then labelled using 6-plex TMT reagents (Thermo Scientific), and subjected to fractionation and purification as previously described . The three biological replicates of CENPK samples were labelled with the 126.1 Th, 127.1 Th and 128.1 Th TMT mass tags, respectively, while the three TDP 1C samples were labelled with the 129.1 Th, 130.1 Th and 131.1 Th TMT mass tags, respectively. After labelling, the six samples were mixed in equal total protein amounts and subjected to a desalting step by means of pre-activated and pre-equilibrated C18 BioPure spin columns (The Nest Group). To reduce sample complexity, TMT-labelled peptides were fractionated by strong cation exchange fractionation (SCX) before liquid chromatography (LC) and MS. Briefly, labelled samples were diluted with 4 volumes of 0.1% formic acid and then applied to the SCX macro spin columns (The Nest Group). After two washings, retained peptides were stepwise eluted in 5 fractions containing increasing concentrations of ammonium formate (i.e., 100, 200, 300, 400, 500 mM). Prior to sample injection into the high-performance liquid chromatography-high resolution tandem mass spectrometry (HPLC-HRMS/MS) workstation, each fraction was desalted by means of the C18 BioPure spin columns. The five labelled fractions of each peptide extract were finally dried using a stream of nitrogen (RT) and suspended in 100 µL of 0.1% formic acid for the subsequent MS analysis.
Relative quantification of yeast proteins was achieved using a hybrid quadrupole-orbitrap Q-Exactive (Thermo Fisher Scientific) mass spectrometer, coupled to a UHPLC system Ultimate 3000 (Thermo Fisher Scientific). Each SCX fraction was separated on a reverse-phase analytical column (Aeris peptide C18, 150 mm × 2.1 mm, 2.6 µm, Phenomenex) kept at 30 °C.
Elution solvents for peptide separation were water (A) and acetonitrile (ACN) (B), both containing 0.1% (v/v) formic acid. The chromatographic separation was carried out at a flow rate of 200 µL ⋅ min− 1 (35 min) using a gradient elution with the following composition (expressed as A:B ratio): 97.5:2.5 for 1 min; from 97.5:2.5 to 70:30 in 19 min (following a linear gradient), from 70:30 to 50:50 in 4 min; from 50:50 to 5:95 in 2 min and maintained for 4 min to wash the column; then back to 97.5:2.5 (to re-equilibrate the system) in 0.5 min and maintained for 4.5 min. The injection volume was 10 µL.
Ion source capillary temperature was 325 °C, the sheath gas flow rate 35 (arbitrary instrument units), auxiliary gas flow rate 10 (arbitrary instrument units), S-lens voltage 55 V, heater temperature 325 °C, and the spray was optimised at 3 kV. The instrument operated in data-dependent mode with a top-7 acquisition method (i.e., a full MS scan at 70,000 resolution on the orbitrap, followed by the MS/MS fragmentation of the seven most intense ions). Full scan spectra were acquired using an automatic gain control (AGC) target of 3 × 106 ions, an injection time (IT) of 250 ms, an isolation window of 2.0 Th, and a scan range from 300 to 2000 Th. Higher energy C‐trap dissociation (HCD) was performed with a NCE of 30, AGC target of 2 ⋅ 105 ions, an IT of 120 ms, a dynamic exclusion of 30 s and a resolution of 17,500. Fragmentation spectra were used for peptide identification and quantification, setting a fixed starting mass of 100 Th. To increase the number of identified peptides, each SCX fraction was analysed twice. To this purpose, the m/z values of peptides positively identified in the first analysis (as described in details in the next section) were used to create a static exclusion list that was then applied to a second HPLC‐HRMS/MS analysis (under the same chromatographic and instrumental conditions) for each sample fraction.
Untargeted MS data analysis.
Raw files derived from HPLC-HRMS/MS runs were analysed with a MudPIT protocol using the Proteome Discoverer 1.4 software (Thermo Fisher Scientific) interfaced to a SEQUEST HT search engine (Thermo Fisher Scientific). All MS/MS data were searched against the UniProt S. cerevisiae database (3AUP000002311). Enzyme specificity was set to trypsin, and a maximum of one missed cleavage was allowed. The precursor and product ion mass tolerances were set to 10 ppm and 0.2 Da, respectively. Oxidation of methionine was selected as variable modification, while 6‐plex TMT at N‐termini and at lysine residues, and carbamidomethylation of cysteines were set as fixed modification. Relative quantification was performed directly by the Proteome Discoverer software and only unique peptides were considered for quantification purposes. Based on the Percolator algorithm, proteins were considered as correctly identified if at least 2 unique peptides were quantified with an individual q-value < 0.05. Proteins were then grouped according to the principle of maximum parsimony. For quantification, the reporter mass tolerance was set to 20 ppm. The software detected the reporter ions (126.1, 127.1, 128.1, 129.1, 130.1, 131.1 Th), and performed the quantification of relative peptide abundance normalizing the intensity of each reporter ion to that of CENPK 1 sample (126.1 Th). Normalized intensity values of proteins derived from Proteome Discoverer were exported in an Excel spreadsheet, and the matrix was arranged for further analysis. The final fold-change of a given protein was calculated as the mean value of the normalized ratios (TDP 1C/CENPK) of the three replicates. Finally, a two-tailed t-test was performed, and only proteins with a ratio > 1.33 or < 0.77, and a p-value < 0.05, were considered over-expressed or under-expressed, respectively.
Parallel reaction monitoring.
The targeted MS-based parallel reaction monitoring (PRM) analysis was carried out in WT CENPK, and TDP-43 1C and 2C strains expressing, or not, NCL. To this end, 50 µg of yeast lysates from nine different cultures for WT CENPK and for each of the 4 different TDP-43 clones (for a total 45 samples) were prepared and processed according to the filter-aided sample preparation (FASP) method as described above. After digestion, peptide mixtures were acidified (pH < 3) by adding formic acid and desalted using BioPure C18 spin columns (The Nest Group), following manufacturer’s instructions. Briefly, samples were loaded in pre-activated C18 spin columns, that were washed twice with 200 µL of 0.5% formic acid (v/v), and then peptides were eluted using 75% ACN containing 0.1% formic acid (v/v). Peptide extracts were dried under a stream of nitrogen and, immediately before HPLC-HRMS/MS analyses, dissolved in a solution (100 µL) containing 5% (v/v) ACN and 0.1% (v/v) formic acid to obtain a final concentration of 0.5 µg ⋅ µL− 1 of protein digest. Confirmatory analyses were performed using the same HPLC-HRMS/MS apparatus described above operating in the PRM mode. Peptides were separated using the same chromatographic gradient described above. The scheduled PRM method was developed by recording and selecting the most intense charge state of the considered peptides and 4 diagnostic precursor-to-product ion transitions in serial injections of a representative yeast digest sample. The same was done for glyceraldehyde 3-phosphate dehydrogenase (GAPDH), which was chosen as housekeeping protein. PRM acquisition parameters were as follows: HCD fragmentation with normalized collision energy (NCE) of 27, AGC target of 5 × 105 ions, an IT of 120 ms, an isolation window of 1.6 Th with an offset of 0.4 Th. Two proteotypic peptides were selected for each protein included in the PRM analysis. The selected precursor-to-product ion transitions, together with the retention time and the instrumental settings used in the acquisition method are reported in Supplemental Table 7.
To ensure that no instrumental drift occurred during PRM analysis, a pooled sample prepared by mixing together equal amounts of peptide digests was injected and analysed at the beginning and at the end of the analytical sequence. The Skyline software (version 220.127.116.1191)  was used to assess the relative abundance of each peptide by calculating the total peak area under the curve (AUC) of the chromatographic peaks deriving from four precursor-to-product ion transitions recorded for target peptides. Each AUC value in the different samples was normalized to the AUC of the corresponding peptide calculated in the pooled sample. Protein abundance was then calculated by averaging the normalized peptide values calculated for the two monitored peptides. Then protein abundance values were further normalized to the GAPDH abundance calculated for each sample to obtain relative protein abundance values to be compared between WT CENPK and the different TDP-43 yeast strains (with or without NCL).
HEK293T cell culture and transfection.
HEK293T cells were grown in Dulbecco’s modified Eagle medium (DMEM) supplemented with 10% (v/v) foetal bovine serum (FBS), 2 mM L-glutamine, 100 U/ml penicillin, and 100 µg/ml streptomycin and maintained at 37 °C in a humidified incubator with 5% CO2. HEK293T cells were seeded at 25% of confluence in multi-well culture plates 24 h before transfection. Cells were (co-)transfected using the Lipofectamine 3000 transfection kit (Invitrogen) following the manufacturer's instructions and analysed 48 h after transfection. Plasmids used to transfect HEK293T cells are reported in Supplemental Table 2. Details regarding the construction of plasmids are described in the Supplemental Methods section.
HEK293T cell viability assay.
Cell viability was assessed using the MTS [3-(4,5‐dimethylthiazol‐2‐yl)‐5‐(3‐carboxymethoxy‐phenyl)‐2‐(4‐sulfophenyl)‐2H‐tetrazolium, inner salt] assay (CellTiter96 Aqueous One 5 Solution Assay, Promega), based on the reduction of MTS by viable cells, following the manufacturer's instructions. Briefly, cells were seeded in 96-wells and co-transfected with the desired plasmids (as indicated in the figure legends), and – 48 h after transfection – the cell culture medium was removed and the MTS reagent [20 µl in 100 µl of phosphate-buffered saline (PBS)] was added to each culture well. Cells were then incubated at 37 °C (90 min), after which the absorbance of reduced MTS (λ = 490) was determined using a Microplate Reader (TECAN) spectrophotometer.
For confocal microscopy observations, HEK293T cells were seeded onto 13 mm coverslips and co-transfected using plasmids coding for the desired fluorescent constructs. 48 h after transfection, cells were rinsed twice with PBS, fixed (30 min, 4 °C) with paraformaldehyde [2% (w/v) in PBS], rinsed again and permeabilized (5 min, 4 °C) with Triton-X100 [0.1% (w/v) in PBS]. Cell nuclei were counter-stained with Hoechst 33342 [(5 µg/ml in PBS) 10 min, RT, Sigma-Aldrich], and coverslips were finally washed in PBS and mounted in Mowiol 40–88 (Sigma-Aldrich) [8% (w/v) in glycerol:PBS (1:3, v/v)]. Images were collected with a Leica SP5 confocal microscope using 40X or 63X HCX PL APO (NA 1.25 or 1.4, respectively) oil-immersion objectives. Laser excitation line, power intensity, and emission range were chosen accordingly to each fluorophore in different samples to minimize bleed-through. During acquisition, parameters for laser intensity and photomultiplier gain were kept constant. Imaging was performed at 1024 × 1024 pixels, with a 200 Hz acquisition rate, by capturing Z-series that covered the entire field of interest. Every planes of a Z-stack scan acquisition were merged into a single image using Fiji/ImageJ software.
HEK293T cell lysis and protein extraction.
For protein extraction, HEK293T cells, grown onto 12-wells plates and transfected as described above, were washed twice with ice-cold PBS and lysed with buffer O (60 µl/well). After centrifugation (14,000 ⋅ g, 10 min, 4 °C) to precipitate cell debris, the total protein content in the supernatant was determined by the BCA assay kit (Thermo Fisher Scientific).
Western blot and antibodies.
For Western blot (WB) analyses, 10–20 µg of proteins diluted in reducing sample buffer (buffer O added with 50 mM DTT and 0.01% (w/v) bromophenol blue ) were subjected to SDS-PAGE [using 10% (w/v) acrylamide-N,N′-methylenebisacrylamide [37.5:1 (w/w)] or Mini-Protean TGX precast gels (4–15%, Bio-Rad Laboratories)] and electroblotted onto polyvinylidene difluoride (PVDF) membranes (0.45 µm pore size; Bio-Rad Laboratories). Membranes were stained with Coomassie brilliant blue (Sigma-Aldrich) to check for even protein loading, and digitalized images were collected for subsequent densitometric analysis. After Coomassie destaining with methanol, membranes were incubated (1 h, RT) in a blocking solution[5% (w/v) non-fat dry milk (Bio-Rad Laboratories) in TRIS-buffered saline (TBS) added with 0.1% (w/v) Tween-20 (TBS-T)], and then probed (overnight, 4 °C) with the desired primary antibody diluted in TBS-T containing 1% (w/v) bovine serum albumin (BSA). After three washings with TBS-T, membranes were incubated (1 h, RT) with horseradish peroxidase-conjugated anti-mouse or anti-rabbit IgG secondary antibody (Sigma-Aldrich, cat. no. A9044 and A0545, respectively) (1:60,000 in blocking solution), depending on the primary antibody. Immunoreactive bands were visualized using an enhance chemiluminescence reagent kit (EMD Millipore) and digitalized by means of an UVItec imaging system (Eppendorf). For densitometric analyses, the intensity of each immunoreactive band was normalized to the optical density of the corresponding Coomassie blue-stained lane .
The following primary antibodies (Abs) were used (dilutions in parentheses): anti-TDP-43 mouse monoclonal (m)Ab (1:1,000, Santa Cruz Biotechnology, cat. no. sc-376532); anti-NCL rabbit polyclonal (p) Ab (1:1,000; Santa Cruz Biotechnology, cat. no. sc-13057); anti GFP mouse mAb (1:2,000, Roche, cat. no.11814460001); anti RFP mouse mAb (1:5,000, Abcam, cat. no. 62341); anti BCL2 rabbit pAb (1:1,000, Sigma-Aldrich, cat. no. SAB4500003)); anti caspase 3 mouse mAb (1:1,000, Santa Cruz Biotechnology, cat. no. sc-56052); anti caspase 9 rabbit pAb (1:1,000, Sigma-Aldrich, cat. no. SAB4300683).
Protein co-immunoprecipitation from S. cerevisiae and HEK293T cell lysates.
For protein co-immunoprecipitation (co-IP) assays from S. cerevisiae, 10 ml of the TDP 2C yeast strain transformed with the NCL-mKATE2-pYES2 construct was grown for 8 h (reaching OD600 ~ 1) in galactose-rich medium, harvested by centrifugation (6,000 x g, 5 min) and resuspended in IP buffer [25 mM Tris-HCl (pH 7), 75 mM NaCl, 1 mM EDTA, 2.5% (w/v) glycerol, 0.2% (w/v) NP-40, 0.5 mM DTT, 1 mM PMSF and complete EDTA-free protease inhibitor cocktail (Roche)]. Cells were lysed by vortexing (30 s, 6000 rpm) in a MagnaLyser® apparatus (Roche Diagnostics). The protein fraction was obtained after precipitation of cell debris by centrifugation (14,000 x g, 10 min, 4 °C), and proteins were quantified by a Lowry assay kit (Sigma-Aldrich). 1 mg of total proteins (in IP buffer) was added with either 1 µg of anti-NCL rabbit pAb (Santa Cruz Biotechnology, cat. no. sc-13057), or anti-GFP mouse mAb (Roche, cat. no. 11814460001), or anti-RFP mouse mAb (Abcam, cat. no. 62341) antibody and incubated (16 h, 4 °C) under continuous gentle inversion mixing. As negative control, the same amount of lysate was incubated in the absence of antibody.
The co-IP protocol for HEK293T cells was adapted from  with minor modifications. Briefly, cells co-transfected with the desired plasmids were resuspended in ice-cold lysis buffer (137 mM NaCl, 2.7 mM KCl, 8 mM Na2HPO4, and 2 mM KH2PO4, 0.2% NP-40, 10% glycerol (w/v), 5 mM EDTA and Roche complete EDTA-free protease inhibitor cocktail), and maintained on ice for 15 min. Cell lysates were then passed (5 times) through 21-gauge needles and centrifuged (20,000 × g, 4 °C, 15 min). The total protein content in the supernatant was determined using the BCA assay kit (ThermoFisher Scientific), and 200 µg of total proteins were incubated (16 h, 4 °C) with 0.2 µg of the rabbit polyclonal anti-NCL (Santa Cruz Biotechnology, cat. no. sc-13057) in lysis buffer under continuous gentle inversion mixing. When needed, cell lysates were treated with RNAse A (Thermo Fisher, cat. no. EN0531, 2 µg, 4 °C, 10 min) prior to immunoprecipitation. Non-transfected cells were processed as described above as negative control.
For both yeast and HEK293 samples, protein-antibody complexes were precipitated by adding protein A-Sepharose (2 mg, Sigma-Aldrich, cat. no. P3391). After incubation (1 h, 4 °C) under gentle shaking, sepharose bead-bound immunocomplexes were collected by centrifugation (3,000 × g, 4 °C), washed three times with lysis buffer and once with 50 mM Tris–HCl (pH 7.5), and finally boiled (3 min) in reducing sample buffer. Immunoprecipitated proteins were separated onto 10% SDS-PAGE gel, electroblotted onto PVDF membranes (Millipore) and analysed by WB with antibodies to target proteins.
Detergent-solubility assays in S. cerevisiae and HEK293T protein extracts.
The protocol for detergent-soluble and -insoluble protein fractionation from yeast cells was adapted from . Briefly, 10 ml of yeast cultures grown for 6 h (reaching OD600 ~ 0.8-1) in inducing (galactose-rich) medium were harvested by centrifugation, washed with water and pelleted again. The cell pellet was resuspended in 500 µL of lysis buffer [25 mM Tris-HCl (pH 7.5), 75 mM NaCl, 1 mM EDTA, 2.5% glycerol (w/v), 0.5% Triton X-100 (v/v), 0.25% deoxycholate (w/v), 0.05% SDS (w/v), 0.5 mM DTT, 1 mM PMSF and complete EDTA-free protease inhibitor cocktail (Roche)]. Cells were lysed by vortexing (30 s ⋅ 6 times, 6,000 rpm) in a MagnaLyser apparatus (Roche Diagnostics), and a small fraction of the crude lysate (“total” fraction) was kept for subsequent analysis. The remaining crude lysate was centrifuged (800 ⋅ g, 5 min, 4 °C) to precipitate cell debris. Fractionation was performed by centrifuging (100,000 x g, 30 min, 4 °C) the cleared lysate, the supernatant (“detergent-soluble” fraction) was saved, and the pellet was resuspended in lysis buffer and centrifuged again as above. The final pellet (“detergent-insoluble” fraction) was resuspended in a urea-based buffer containing 7 M urea, 2 M thiourea, 4% CHAPS and 30 mM Tris (pH 8.5).
Detergent solubility fractionation of proteins from HEK293T cells was performed according to  with some modifications. Cells grown in 6-well plates and transfected as previously described were washed in PBS and harvested by centrifugation (2,000 ⋅ g, 5 min, 4 °C). Cells were then resuspended in 300 µL of radio-immunoprecipitation assay (RIPA) buffer [20 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1% NP-40 (v/v), 0.1% sodium deoxycholate (w/v), 1 mM Na3VO4] supplemented with 2 mM EDTA, 1 mM EGTA and complete EDTA-free protease inhibitor cocktail (Roche). Following incubation on ice (15 min), cells were sonicated (10 s) and the obtained lysate was cleared by centrifugation (500 ⋅ g, 10 min) to remove cell debris. A small amount of the lysate (“total” fraction) was kept for subsequent analysis, while the remaining lysate was centrifuged (100,000 ⋅ g, 30 min, 4 °C). The resulting supernatant (“detergent-soluble” fraction) was saved and the pellet, after washing in RIPA buffer, was centrifuged again as above. The final pellet (“detergent-insoluble” fraction) was resuspended in the same urea-based buffer used for yeast samples.
The quantification of the protein content in all fractions of both yeast and HEK293T samples was carried out by the Lowry assay kit (Sigma-Aldrich). Protein fractions were then analysed by SDS/PAGE (using 10% (w/v) acrylamide-N,N′-methylenebisacrylamide [37.5:1 (w/w)) and WB for the detection of the target proteins as described previously.
The protein–protein interaction network and gene ontology enrichment analysis was performed using the publicly available Cytoscape 3.7 software  integrated with the STRING application . The STRING database uses a combination of prediction approaches integrated with other information (i.e., neighbourhood, transferred neighbourhood, gene fusion, co-occurrence, co-expression, experimental data, databases, text mining). The protein network was built using default parameters (minimum required interaction score = 0.4), including all active prediction parameters.
Data were analysed using Prism 7 (GraphPad Software) and Microsoft Excel 2013 (Microsoft Corporation) software. Data are reported as mean ± standard error of the mean (SEM), with the number of experimental replicates (n) being indicated in the figure legends. Statistics was based on unpaired two-tailed Student’s t-test, or by Kruskal-Wallis test followed by a Dunn's post hoc test, depending on the experiment, as indicated in the figure legends. A p-value < 0.05 was considered statistically significant (* p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001).