Cell culture

HEK293T and U2OS cells were obtained from ATCC to create all cell lines. Cells were maintained in Dulbecco’s DMEM with 10% fetal bovine serum, 1% penicillin–streptomycin, and 1% GlutaMax. Unless otherwise mentioned, v2L tau biosensor cells were used for all seeding assays. Details on these biosensors have been recently published [8]. Cell lines were checked for mycoplasma contamination (Venor-GEM Mycoplasma Detection kit).

Proteomics screen

T225 flasks were coated with 10 mL of 0.01 mg/mL poly-D-lysine (PDL) for 3 h in the incubator and rinsed with PBS before plating cells. 22 million cells were plated in 25 ml/T225 flask and allowed to settle overnight. The following day, cells were treated with 50 nM tau + Lipofectamine-2000 complexes (or 50 nM α-synuclein + Lipofectamine as a negative control) which were incubated for 20 min at RT prior to addition to cells. Cells were incubated with the fibrils for 5 h. Thirty minutes before the 5 h time point, cells were treated with BP (biotin phenol) at a final concentration of 500 µM at 37ºC. At 5 h, cells were treated with H2O2 at a final concentration of 1 mM and the flasks were agitated at RT for 1 min. The biotinylation reaction was quenched with the quenching buffer followed by three additional rinses. Quenching buffer was also used to scrape the cells to collect the cell pellets. This buffer was prepared as previously described in the APEX2 labeling protocol [68].

On-Bead trypsin digestion

Protein concentrations were normalized across all the samples (~ 1 mg of starting lysate) based on the Pierce 660 assay readings and protein abundances from shotgun proteomics analysis of trypsin digests of these samples by the UT Southwestern Proteomics Core Facility. Lysates (1 mg) were incubated with 250µL of magnetic streptavidin beads at 4ºC for overnight incubation ~ 16 h. The next day, the beads were concentrated by magnet, and washed 2 × with 200µL of 50 mM Tris–HCl pH 7.5 followed by 2 × with 2 M urea + 50 mM Tris–HCl pH 7.5. The beads were then incubated with 80µL 2 M urea + 100μL 0.5ug/µl trypsin + 20µL 10 mM DTT to achieve a final urea concentration of 1 mM and a ratio of 1: 20 for trypsin: lysate, for 1 h at 25ºC with shaking at 1000 rpm in a thermomixer. The beads were washed 2x with 60 µl of 2 M urea + 50 mM Tris–HCl pH 7.5 and the two washes were combined with the supernatant. The eluate was reduced with DTT at a net concentration of 4 mM by incubating for 30 min with shaking at 1000 rpm, 25ºC. The samples were alkylated with 10 mM iodoacetamide for 45 min at 25ºC with shaking at 1000 rpm.

50 mM Tris–HCl pH 7.5 was then added to achieve a final urea concentration of 0.73 M. Samples were incubated overnight (~ 15 h) at 37ºC with shaking at 1000 rpm to allow complete trypsin digestion. The samples were removed from the thermomixer and spun down. Trypsin was quenched by acidifying the samples to pH < 3 with the addition of formic acid at a final concentration of 1%.

TMT Mass spectrometry

5μL of 10% trifluoroacetic acid (TFA) was added to each sample, and solid-phase extraction was performed on each sample using an Oasis HLB 96-well μElution plate (Waters). Eluates were dried and reconstituted in 50μL of 100 mM triethylammonium bicarbonate (TEAB). 10μL of each sample was labeled with 4μL of a different TMT10plex reagent (Thermo Scientific, label TMT10-131 not used). Samples were quenched with 1μL of hydroxylamine, mixed, and dried in a SpeedVac. Samples were reconstituted in 2% acetonitrile, 0.1% formic acid to a concentration of 0.5 ug/μL.

2μL of each TMT sample were injected onto an Orbitrap Fusion Lumos mass spectrometer coupled to an Ultimate 3000 RSLC-Nano liquid chromatography system. Samples were injected onto a 75 μm i.d., 75-cm long EasySpray column (Thermo) and eluted with a gradient from 0–28% buffer B over 180 min. Buffer A contained 2% (v/v) acetonitrile (ACN) and 0.1% formic acid in water, and buffer B contained 80% (v/v) ACN, 10% (v/v) trifluoroethanol, and 0.1% formic acid in water. The mass spectrometer was operated in positive ion mode with a source voltage of 1.8 kV and an ion transfer tube temperature of 275 °C. MS scans were acquired at 120,000 resolution in the Orbitrap and top speed mode was used for SPS-MS3 analysis with a cycle time of 2.5 s. MS2 was performed with CID with a collision energy of 35%. The top 10 fragments were selected for MS3 fragmentation using HCD, with a collision energy of 55%. Dynamic exclusion was set for 25 s after an ion was selected for fragmentation.

Proteomics data analysis

Raw MS data files were analyzed using Proteome Discoverer v2.4 (Thermo), with peptide identification performed using Sequest HT searching against the human protein database from UniProt. Fragment and precursor tolerances of 10 ppm and 0.6 Da were specified, and three missed cleavages were allowed. Carbamidomethylation of Cys and TMT labeling of N-termini and Lys sidechains were set as a fixed modification, with oxidation of Met set as a variable modification. The false-discovery rate (FDR) cutoff was 1% for all peptides.

For every biological replicate, absolute abundance of each protein was first normalized to the total protein abundance of a particular lysate sample to account for any differences in total protein concentrations across samples before comparison. These values were used to calculate the relative enrichment of proteins specific to tau seeding: Protein Abundance Ratio = (sAPEX2 P301S + seeds)/(sAPEX2 P301S – seeds).

Normalized protein abundance ratios for sAPEX2 P301S and sAPEX2 alone (negative control) treated with and without tau fibrils were compared using unpaired t- test on three independent biological replicates; two-stage step-up (Benjamini, Krieger, and Yekutieli), FDR 1.00%). Positive relative abundance values on the graph indicate enrichment in the aggregation proteome. Statistical significance was determined based on q value. In the dataset, MAPT appears as a negatively enriched relative abundance value due to background signal from the sAPEX2 alone ratios (+ seeds /- seeds). This artifactual result was thus not displayed on the volcano plot.

Generation of a WT tau biosensor cell line

FM5 CMV promoter plasmids containing the mCerulean3 and mRuby3 fluorophores were digested with Esp3I restriction enzyme. The 3R and 4R isoforms of WT tau aa 246–408 were cloned into the cut plasmids using Gibson assembly, respectively. Constructs were sequence verified using UTSW’s Sanger sequencing core and used for making lentivirus. HEK293T cells were transduced with lentivirus containing both constructs for 48 h after which single cells expressing both fluorophores were sorted into 96-well plates. Monoclonal lines were tested for lipofectamine-mediated seeding with AD brain homogenate and the most sensitive clone was selected.

Generation of a TDP-43 biosensor cell line

FM5 CMV promoter plasmids containing the mClover3 and mRuby3 fluorophores were digested with Esp3I restriction enzyme. WT TDP-43 (aa 275–414 with the addition of an alanine after the start site and preceding the actual TDP43 sequence) was cloned into the cut plasmids using Gibson assembly to generate constructs expressing TDP-43 tagged to the respective fluorophores. Constructs were sequence verified using UTSW’s Sanger sequencing core and used for making lentivirus.

HEK293T cells were transduced with the TDP-43 lentivirus and were sorted into monoclonal cells for highest expression of the two fluorophores. Monoclonal cell line that responded with maximum seeding to different FTLD TDP-43 lysates was chosen for the seeding assay reported here. Cells expressing the individual fluorophores were also sorted as fluorophore compensation controls for detecting FRET on the flow cytometer.

Biosensor seeding assay

All biosensor assays were performed with “naked seeding” (no cation-based transfection reagent). Cell lines were plated at a density of 15,000 cells/well of a 96 well plate and allowed to settle overnight. Cells were treated with an appropriate concentration of recombinant tau fibrils for 48 h, and then harvested for flow cytometry. Fibril preps were sonicated in a water bath sonicator for 1 min at 65Amp prior to cell treatment. Recombinant tau fibrils were prepared as previously described [28].

For seeding with brain lysates and for the α-synuclein biosensor seeding assay, 8,000 cells/well (10,000 cells/well for TDP-43 biosensors) were plated in 96 well plates and seeding was monitored for 72 h (or 5 days for WT tau biosensors). In the case of brain homogenates, biosensors were treated with 25 µg of tauopathy lysates and 10 µg of FTLD lysate. These were sonicated for 1 min at 65 Amp in a water bath sonicator. For seeding with α-synuclein, fibrils were sonicated for 5 min total, 1 min on /1 min off at 65 Amp, and used at a final concentration of 400 nM.

All seeding results are reported as % FRET + cells. The FRET data were plotted by subtracting the background signal (no exogenous tau added) which was negligible for all conditions (no FRET recorded in the absence of tau seeds), unless otherwise specified.

Brain homogenization

Brain tissue from clinically and neuropathologically characterized cases of AD, CBD, and FTLD TDP-43 were obtained from UTSW and Washington University in St. Louis. All human tissues used in these experiments were derived from autopsy subjects. Since deceased subjects are not considered human subjects for research purposes, these studies were exempt from human subjects research regulations and did not require IRB approval. Brain samples were weighed and added to 1X TBS supplemented with cOmplete Ultra (Roche) protease inhibitor to prepare a 10% w/v solution. The brains were homogenized using a probe homogenizer to obtain a slurry that was sonicated for 15 min total, 1 min on/ 30 s off. The sonicated samples were centrifuged at 4C for 15 min at 21,300 × g. Protein concentration of the supernatant was measured using Pierce 660 assay and was subsequently used for naked seeding.

Uptake assay

The uptake assay was performed as previously described [28]. v2L cells were plated overnight at a density of 8,000 cells/well of a 96 well plate. Cells were treated with 25 nM of AF-647 labeled tau fibrils or AF-647 dye alone as a negative control. After 4 h of incubation with the fibrils, cells were harvested with 0.25% trypsin for flow cytometry.

The labeled fibrils used in this assay were obtained by incubating recombinant tau fibrils (8 μM, 200μL) with lyophilized AF-647 dye (25 μg) for 1 h at room temperature (RT) followed by quenching the reaction with 100 mM glycine and subsequent dialysis in a 3500 kDa dialysis cassette.

The median fluorescence intensity (MFI) values representing the amount of tau internalized were plotted after subtracting the background MFI of the dye alone signal for all conditions. These MFI values were then normalized relative to the appropriate control condition of the respective experiment (DMSO, NTG, or Scr controls).

Compound treatments

96 well plates were coated with 0.01 mg/mL PDL and incubated at 37C for 3 h followed by washout with PBS. v2L cells were plated at a density of 15,000 cells/well and allowed to settle overnight. Cells were treated with different compounds (ML-240, NMS-873, MG132, and LLOMe) for about an hour upon which 25 nM of recombinant tau fibrils were introduced to the media. After four hours of incubation with the fibrils (five hours with compounds), the media was replaced with fresh media and uptake or seeding was monitored for 4 h and 48 h, respectively.

Flow cytometry

To harvest cells for flow cytometry, media was removed, and cells were treated with 0.05% trypsin (0.25% trypsin for uptake assay) for 5 min at 37C (0.25% trypsin, 15 min at 37C in case of PDL coated plates). Trypsin was quenched with cold media and cells were resuspended a few times before transferring the suspension to 96-well round-bottom plates which were centrifuged at 1000 rpm for 5 min. Supernatant was removed and the cell pellets were resuspended in 2% paraformaldehyde (PFA) and allowed to fix for 10 min at RT. Cells were spun down again, PFA was removed, and cells were resuspended in PBS and stored at 4C until ready to be run on a flow cytometer (BD LSRFortessa) for quantifying the FRET signal.

Cloning

FM5 vector with UBC promoter was used to clone all the APEX constructs. sAPEX fragments (AP and EX) were PCR amplified from the constructs provided by the laboratory of Alice Ting (Stanford). Amplified sAPEX fragments were appended on to the c-terminus of RD tau fragments via a linker using overlap PCR. Using Gibson assembly, the final gene fragments were cloned into FM5 UBC plasmid which was cut with Esp3I. All Gibson reaction products were transformed into Stbl3 bacterial cells. Bacterial colonies were inoculated, DNA was purified using Qiagen miniprep kit, and the sequences were verified using Sanger sequencing at UTSW’s sequencing facility.

Lentivirus production

Low passage HEK293T cells were plated at ~ 70% confluency in 6 well plates and allowed to settle overnight. A master mix was prepared using 400 ng of plasmid of interest, 400 ng of VSVG, and 1200 ng of PSP plasmids required for virus packaging, along with 7.5L of TransIT 293 and 120ul of OMEM per well of a 6 well plate. The master mix was allowed to incubate at RT for 30 min upon which it was added to the cells drop-wise. The virus was harvested 48 h later by collecting the media, centrifuging it for 5 min at 1000 rpm, and then freezing the aliquoted supernatant.

Differentiation and culturing of human iPSC-derived cortical neurons

We utilized the integrated, inducible, and isogenic Ngn2 iPSC line (i3N). It was previously shown that expression of the transcription factor neurogenin-2 (Ngn2) induces rapid differentiation of iPSCs into cortical glutamatergic neurons [69]. This iPSC line harbors a doxycycline-inducible mouse Ngn2 transgene at an adeno-associated virus integration site 1 (AAVS1) safe-harbor locus, allowing for a simplified differentiation protocol [70, 71]. iPSCs were dissociated with Accutase (Sigma, A6964) and plated onto basement membrane extract-coated plates (R&D, 3434–001–02). Ngn2 expression was induced with 2 μM doxycycline hyclate (Sigma, D9891) in KSR media alone with 10 μM SB431442 (R&D, 1614), 2 μM XAV939 (Stemgent, 04–0046) and 100 nM LDN-193189 (Stemgent, 04–0074) (doxycycline hyclate is maintained in all medias going forward). On day 2, cells were fed with a 1:1 ratio of KSR media + SB/XAV/LDN and N2-supplmented neural induction media with 2 μg/ml puromycin (Life Technologies, A1113803). On day 3, cells were fed with N2-supplemented neural induction media. On day 4, cells were dissociated with Accutase and plated onto PDL-(Sigma, P1149) and laminin-(Life Technologies, 23 017–015) coated tissue culture plates. Cells were subsequently maintained with neurobasal media (Life Technologies, 21,103,049) supplemented with NeuroCult SM1 (StemCell Technologies, 05711) and 10 ng/ml brain-derived neurotrophic factor (R&D, 248-BD-005/CF) until collected.

Compound treatment and seeding assay in neurons

Differentiated human neurons were treated with tau RD P301S lentivirus (tau-clover at an MOI of 3; tau-ruby at an MOI of 2). 48 h later, the transduced cells were simultaneously incubated with 15 nM FL, WT recombinant tau fibrils and the VCP inhibitors, ML-240 and NMS-873 at 100 nM for 48 h. For acute exposure with tau fibrils and the compounds, the neuronal biosensors were treated with 1 μM ML-240 and 100 nM NMS-873 for 4 h followed by media replacement. FRET signal, used as a measure of tau aggregation, was monitored over 48 h using ImageXpress Confocal HT.ai High-Content Imaging System (Molecular Devices). 4 images per well, with 5 wells per condition of a 96 well plate, were collected for the clover, ruby, and FRET channels. Single color controls were used to correct for fluorophore bleed through using an automated algorithm on MATLAB. Single color controls were used to measure the bleedthrough coefficient of each channel by fitting the correlation of pixel intensities in the single color channel and the “empty” channel with a linear fit. Images from the acceptor/donor channels of real data were then multiplied by the resulting slopes (i.e. bleedthrough coefficients) to approximate the amount of bleedthrough. Finally, FRET channel images were corrected by subtracting these bleedthrough images from the FRET images. The corrected images were analyzed to calculate the FRET area using an ImageJ macro and the quantified FRET area was plotted for different conditions.

Imaging

All high magnification images were taken on the Nikon SoRA at 60x (oil immersion) magnification. For imaging, 96-well Cellvis glass bottom plates were coated with PDL followed by washout before plating cells of interest. Nuclei were stained with Hoechst (1 g/mL) for 10’ at 37C prior to fixing cells in 4% PFA for 10’ at RT. Cells were washed twice with PBS, 5’ each, and saved in PBS at 4C until imaged on the microscope.

CRISPR/Cas9 Screen for VCP Cofactors

CRISPR constructs for the cofactors were outsourced to Twist Biosciences for synthesis. Constructs not synthesized by the company were cloned in the lab using standard ligation reactions. Four guides per gene were chosen from the Brunello library deposited online and ordered as duplex DNA from IDT. LentiCRISPRv2 (Addgene #52961) was cut using Esp3I, and T4 ligase was used for all ligation reactions of the guides into the plasmid. Stbl3 bacteria were transformed with the ligated products, selected colonies were inoculated, mini-prepped using Qiagen kit, and the purified DNA was sequence-verified. Pooled lentivirus was prepared with four constructs per gene and v2L cells were transduced with the virus at the desired MOI. After 24 h, cells were expanded in puromycin media (2μg/mL) for selection of the KO population. Selected populations were eventually used for seeding and uptake assays. The KO was verified using western blot.

siRNA Knockdown

siRNAs were ordered from Origene. 300,000 v2L cells were plated in 6 well plates and allowed to settle overnight. The next day, cells were treated with 100 nM of each siRNA, with a total of three siRNAs per gene using RNAiMax Lipofectamine (Thermo) as a transfection vehicle at 7.5 µl/well. After 48 h of transfection, the cells were plated in 96 well plates for seeding and uptake assays and used for western blot to verify the knockdown.

Western blot

Cell pellets were lysed in RIPA buffer and allowed to sit on ice for 5 min followed by a 15,000 g spin for 10 min at 4C. The supernatants were used to determine the protein concentrations using Pierce 660 assay. 15 μg of total protein was treated with SDS buffer + BME and heated at 95C for 10 min. Samples were loaded onto 4–12% bis–tris gels and the proteins were transferred onto nitrocellulose membranes using the Biorad turbo transfer machine. All incubations for subsequent steps were done in TBS + 0.05% Tween-20 (TBST). The membranes were first incubated in blocking buffer (5% milk powder + TBST) for 1 h at RT, followed by primary antibodies in the blocking buffer at 4C with overnight shaking. After the primary antibody incubation, the membranes were washed 3x with TBST, 10 min each. Then, appropriate HRP-conjugated secondary antibodies in blocking buffer were added to the membranes for a 1.5 h incubation at RT. Membranes were again washed 3 × in TBST followed by a single wash in TBS alone before reading the HRP signal using the Thermo ECL kit.

Statistical analysis

Proteomics data was analyzed using unpaired t- test, two-stage step-up (Benjamini, Krieger, and Yekutieli), FDR 1.00%. One-Way ANOVA (Šídák method) with a 95% confidence interval was used for all the other statistical analyses unless otherwise stated. Paired t-test was used to analyze the neuron data.

For all seeding experiments, one representative experiment of three biological replicates is presented. Each experiment includes technical triplicates. Thus, the seeding data shows S.D. Uptake assay data was normalized across all three biological replicates and is therefore presented with S.E.M.

The P values are described as follows: ns = not significant/ P > 0.05, * = P ≤ 0.05, ** = P ≤ 0.01, *** = P ≤ 0.001, **** = P ≤ 0.0001.

Graphics

Biorender.com was used to create the graphics presented here and has granted publication and licensing rights (agreement number: RX26PLQVWQ).

List of Reagents

Reagent

Vendor

Catalog No

Acetonitrile ≥ 99.9%, LC–MS Reagent for LC–MS, for HPLC

Avantor

9829–03

Ammonium bicarbonate,ReagentPlus®, ≥ 99.0%

Sigma-Aldrich

A6141-500G

Anti-FAF2 Rabbit Polyclonal Antibody, Size = 150 µL

Fisher Scientific

16,251–1-AP

Anti-OTUB1 antibody [EPR13028(B)] (ab175200)

Abcam

ab175200

Anti-UBE4B antibody [EPR7471] (ab126759)

Abcam

ab126759

Anti-UBXD7 Antibody

EMD Millipore

AB10037

Anti-VCP antibody (ab11433)

Abcam

ab11433

Ataxin 3 Antibody

Fisher Scientific

702,788

ATXN3 (Human)—3 unique 27mer siRNA duplexes—2 nmol each

OriGene

SR302905

Benzonase Nuclease, ≥ 250 units/muL, ≥ 90% (SDS-PAGE)

Sigma-Aldrich

E1014-5KU

Biotinyl Tyramide, Tocris, 6241

R&D Systems

6241/25

Biotinyl tyramide, ≥ 97% (HPLC)

Sigma-Aldrich

SML2135-50MG

BME, ≥ 99.0%

Sigma-Aldrich

M6250-100ML

96 well glass bottom plates

Cellvis

P96-1.5H-N

Complete™, Mini, EDTA-free Protease Inhibitor Cocktail

Sigma-Aldrich

4,693,159,001

Corning 225cm2 Angled Neck Cell Culture Flask with Vent Cap

Corning

431,082

FAF2 (Human)—3 unique 27mer siRNA duplexes—2 nmol each

OriGene

SR308083

Formic acid 50ML UN 1779 3(8) / PGII

Sigma-Aldrich

56,302-50ML-GL

GAPDH Antibody (1D4)

Fisher Scientific

NB300-221

Gibson Assembly Master Mix

New England Biolabs

E2611S

Hoechst 33,342

Thermo Scientific

H3569

Invitrogen novex NuPAGE 4 12% Bis Tris Protein Gels, 1.0 mm, 10 well

Thermo Scientific

NP0321BOX

Iodoacetamide,single use vial of 56 mg

Sigma-Aldrich

A3221-10VL

Iproof™ High-Fidelity PCR Kit, 200 U (2 U/µl), 100 µl 1,725,331

Bio-Rad

1,725,331

Jumpstart™ Taq DNA Polymerase,with MgCl2

Sigma-Aldrich

D9307-50UN

Laemmli Sample Buffer 2X

Bio-Rad

1,610,737

Laemmli Sample Buffer, 4X

Bio-Rad

1,610,747

Lipofectamine RNAiMAX Transfection Reagent

Fisher Scientific

13–778-075

MG-132 25 mg

Fisher Scientific

S2619

Ml240, ≥ 98% (hplc)

Sigma-Aldrich

SML1071-5MG

NEBuilder HiFi DNA Assembly Master Mix—10 reactions

New England Biolabs

E2621S

NGLY1 (Human)—3 unique 27mer siRNA duplexes—2 nmol each

OriGene Technologies

SR310927

NGLY1 Polyclonal Antibody

Thermo Scientific

A305-547A-T

NheI-HF® Restriction Enzyme

New England Biolabs

R3131S

NMS-873

MedChem Express

HY-15713

Npl4 Antibody

Cell Signaling Technology

13489S

NPLOC4 (Human)—3 unique 27mer siRNA duplexes—2 nmol each

OriGene

SR310841

NSFL1C (Human)—3 unique 27mer siRNA duplexes—2 nmol each

OriGene

SR311050

Nsfl1c Polyclonal Antibody

Fisher Scientific

PA5-21,633

Nupage™ 4 12% Bis Tris Protein Gels, 1.5 mm, 15 well

Fisher Scientific

NP0336BOX

Nupage™ 4–12% Bis–Tris Protein Gels, 1.5 mm, 10-well

Fisher Scientific

NP0335BOX

One Shot Stbl3 Chemically Competent

Thermo Scientific

C737303

Opti-MEM™ I Reduced Serum Medium

Fisher Scientific

31–985-070

Opti-MEM™ I Reduced Serum Medium

Thermo Fisher Scientific

31,985,070

Pierce™ 660 nm Protein Assay

Fisher Scientific

22,660

Pierce™ BCA® Bovine Serum Albumin Standard Set

Thermo Scientific

23,208

Pierce™ Nitrocellulose Membranes, Thermo Scientific, Roll

Fisher Scientific

88–018

Pierce™ Streptavidin Magnetic Beads

Thermo Fisher Scientific

88,817

PLAA (Human)—3 unique 27mer siRNA duplexes—2 nmol each

OriGene

SR306209

Poly-D-lysine hydrobromide,mol wt 70,000–150,000, lyophilized powder

Sigma-Aldrich

P6407-5MG

Precision Plus Protein™ Dual Color Standards, 10–250 kDa

Bio-Rad

1,610,374

QIAprep Spin Miniprep Kit (250)

Qiagen

27,106

Redtaq® ReadyMix™ PCR Reaction Mix,Complete PCR reagent

Sigma-Aldrich

R2523-100RXN

RPS27A Human siRNA Oligo Duplex (Locus ID 6233)

OriGene

SR304187

S.O.C. Medium

Thermo Fisher Scientific

15,544,034

Sequencing Grade Modified Trypsin, Promega

Promega

V5111

Sodium Ascorbate, Powder, USP, Packaging = Poly Bottle, Size = 100 g

Spectrum Chemical

S1349-100GM

SVIP (Human)—3 unique 27mer siRNA duplexes—2 nmol each

OriGene

SR316907

SYVN1 Human siRNA Oligo Duplex (Locus ID 84447)

OriGene

SR325336

T4 DNA Ligase

New England Biolabs

M0202L

Thermo Scientific Pierce DTT (Dithiothreitol)

Thermo Fisher Scientific

20,290

Thermo Scientific 6X DNA Loading Dye

Thermo Fisher Scientific

R0611

Thermo Scientific FastDigest BsmBI (Esp3I) Promotion

Thermo Fisher Scientific

FD0454

Thermo Scientific Pierce 660 nm Protein Assay

Fisher Scientific

PI22660

Trans-Blot, 1,704,270

Bio-Rad

1,704,270

Trans-Blot® Turbo™ RTA Midi Nitrocellulose Transfer Kit, for 40 blots

Bio-Rad

1,704,271

TransIT®−293 Transfection Reagent

Fisher Scientific

MIR 2700

Trolox

Sigma-Aldrich

238,813-5G

Tween® 20, viscous liquid, CAS 9005–64-5, Sigma-Aldrich P1379-1L

Sigma-Aldrich

P1379-1L

UBE4B (Human)—3 unique 27mer siRNA duplexes—2 nmol each

OriGene

SR306958

UBXN6 (Human)—3 unique 27mer siRNA duplexes—2 nmol each

OriGene

SR312922

UBXN6 Polyclonal Antibody

Thermo Scientific

PA5-84,520

Ufd1 Antibody

Cell Signaling Technology

13789S

UFD1L (Human)—3 unique 27mer siRNA duplexes—2 nmol each

OriGene

SR305021

VCP (Human)—3 unique 27mer siRNA duplexes—2 nmol each

OriGene

SR322176

Venor™ GeM Mycoplasma Detection Kit, PCR-based

Sigma-Aldrich

MP0025-1KT

Vinculin Antibody

Fisher Scientific

NBP2-41,237