HUWE1 controls tristetraprolin proteasomal degradation by regulating its phosphorylation
- Department of Microbiology, Immunobiology and Genetics, Max Perutz Labs, University of Vienna, Vienna BioCenter (VBC), Austria
- Vienna BioCenter PhD Program, Doctoral School of the University of Vienna and Medical University of Vienna, Vienna BioCenter (VBC), Austria
- Research Institute of Molecular Pathology (IMP), Vienna BioCenter (VBC), Austria
- Medical University of Vienna, Vienna BioCenter (VBC), Austria
Figures
Figure 1 with 1 supplement
TTP is degraded by the proteasome in a ubiquitin-dependent manner.
(A) RAW264.7 murine macrophages were stimulated with LPS and incubated with the translation inhibitor cycloheximide (CHX) and the proteasome inhibitor MG132 for the indicated times (h), after which …
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Figure 1—source data 1
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Figure 1—source data 2
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Figure 1—source data 3
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Figure 1—source data 4
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Figure 1—source data 5
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Figure 1—figure supplement 1
TTP is degraded by the proteasome in a ubiquitin-dependent manner.
(A) 3xHA-TTP-expressing RAW264.7 cells were incubated with LPS or left unstimulated. Cells were then treated with E1 enzyme inhibitor (TAK-234) or the proteasome inhibitor Epoxomicin (Epx). …
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Figure 1—figure supplement 1—source data 1
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Figure 1—figure supplement 1—source data 2
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Figure 1—figure supplement 1—source data 3
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Figure 1—figure supplement 1—source data 4
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Figure 1—figure supplement 1—source data 5
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Figure 1—figure supplement 1—source data 6
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Figure 1—figure supplement 1—source data 7
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Figure 1—figure supplement 1—source data 8
TurboID-TTP proximity labelling in RAW264.7 cells.
- https://cdn.elifesciences.org/articles/83159/elife-83159-fig1-figsupp1-data8-v1.zip
Figure 2 with 1 supplement
Genome-wide CRISPR-Cas9 knockout screen identified the E3 ligase HUWE1 as a regulator of TTP stability.
(A) Overview of FACS-based CRISPR-Cas9 knockout screening procedure using the RAW264.7-Dox-Cas9-mCherry-TTP cell line. Cells expressing high and low levels of mCherry-TTP protein were sorted, and …
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Figure 2—source data 1
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Figure 2—source data 2
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Figure 2—source data 3
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Figure 2—source data 4
Cas9 functionality and leakiness evaluation.
RAW264.7-Dox-Cas9 cells expressing an sgRNA targeting the cell-essential gene Rrm1 or the ROSA safe-harbor locus. sgRNA-positive cells were monitored by flow cytometry in the presence or absence of Dox over the indicated time period. Relative cell viability of sgRrm1-transduced cells was compared to untransduced cells, normalized to sgROSA relative cell viability and plotted.
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Figure 2—source data 5
Adopted gating strategy of FACS-based mCherry-TTP stability regulator screen.
Representative scatter plots are shown in hierarchical order. First debris, doublets, dead (APC-Cy7 positive), Cas9-negative (GFP), mCherry- and BFP-negative cells were excluded. 5% of cells with the lowest and 1% of cells with the highest mCherry and BFP signal were sorted.
- https://cdn.elifesciences.org/articles/83159/elife-83159-fig2-data5-v1.zip
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Figure 2—source data 6
MAGeCK analysis of D3 and D6 CRISPR screen in RAW-Dox-Cas9.
- https://cdn.elifesciences.org/articles/83159/elife-83159-fig2-data6-v1.zip
Figure 2—figure supplement 1
Genome-wide CRISPR-Cas9 knockout screen identified the E3 ligase HUWE1 as a regulator of TTP stability.
(A) Read counts per million in the mCherry-TTPhigh cells at 6 days after Cas9 induction were compared to those in unsorted cells from the same day, sgRNA enrichment calculated by MAGeCK analysis, …
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Figure 2—figure supplement 1—source data 1
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Figure 2—figure supplement 1—source data 2
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Figure 2—figure supplement 1—source data 3
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Figure 3 with 1 supplement
TTP mRNA targets are dysregulated upon Huwe1 depletion.
RAW264.7-Dox-Cas9 cells expressing sgROSA or sgHuwe1 were treated with Dox for 5 days to induce Cas9. Cells were incubated with LPS for the indicated time points (h). (A) Mature Tnf mRNA levels, and …
Figure 3—figure supplement 1
TTP mRNA targets are dysregulated upon Huwe1 depletion.
RAW264.7-Dox-Cas9 cells expressing sgROSA or sgHuwe1 were treated with Dox for 5 days to induce Cas9. Cells were incubated with LPS for the indicated time points (h). (A) Tnf or (B) Il6 mRNA levels …
Figure 4 with 1 supplement
HUWE1 regulates TTP phosphorylation status, and thereby TTP stability.
(A–D) RAW264.7-Dox-Cas9 cells expressing sgROSA or sgHuwe1 were treated with Dox for 5 days to induce Cas9. Cells were incubated with LPS for the indicated time points (h). Phosphorylation of (A) …
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Figure 4—source data 1
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Figure 4—source data 2
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Figure 4—source data 3
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Figure 4—source data 4
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Figure 4—source data 5
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Figure 4—figure supplement 1
HUWE1 regulates TTP phosphorylation status, and thereby its TTP stability.
(A–D) RAW264.7-Dox-Cas9 cells expressing sgROSA or sgHuwe1 were treated with Dox for 5 days to induce Cas9. Cells were incubated with LPS for the indicated time points (min). Phosphorylation of (A) …
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Figure 4—figure supplement 1—source data 1
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Figure 4—figure supplement 1—source data 2
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Figure 4—figure supplement 1—source data 3
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Figure 4—figure supplement 1—source data 4
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Figure 4—figure supplement 1—source data 5
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Figure 5 with 1 supplement
TTP family member ZFP36L1 abundance is increased upon HUWE1 knockout in human and mouse cells.
(A) RAW264.7-Dox-Cas9 expressing sgROSA, sgHuwe1 or sgPsmb7 were treated with Dox for 3 days to induce Cas9. Proteome changes were assessed by quantitative mass spectrometry. Proteins classified as …
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Figure 5—source data 1
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Figure 5—source data 2
Quantitative proteomics to systematically assess protein changes after HUWE1 knockout, in RAW264.7-Dox-Cas9, A375, and SW620 cell lines.
- https://cdn.elifesciences.org/articles/83159/elife-83159-fig5-data2-v1.zip
Figure 5—figure supplement 1
Abundance of the TTP family member ZFP36L1 is increased upon HUWE1 knockout in human and mouse.
(A) Proteome changes in RAW264.7-Dox-Cas9 cells expressing sgPsmb7 by quantitative mass-spectrometry. Proteins classified as PSMB7 targets are highlighted in orange and displayed in the Huwe1 …
Figure 6 with 1 supplement
Residues in the TTP 234–278 region are important for its stability.
(A) Schematic representation of 3xHA-TTP mutants. Colors denote amino acid substitutions. ZF indicates the zinc finger domain, and the three tetraprolin motifs are presented as dark grey boxes. (B–E)…
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Figure 6—source data 1
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Figure 6—source data 2
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Figure 6—source data 3
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Figure 6—source data 4
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Figure 6—figure supplement 1
Residues in the TTP 234–278 region are important for its stability.
(A) Alignment of mouse and human TTP and ZFP36L1 orthologs. (B) TTP protein levels from Figure 6B were quantified from non-saturated western blot signals, normalized to the internal control mCherry, …
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Figure 6—figure supplement 1—source data 1
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Figure 6—figure supplement 1—source data 2
Blots corresponding to Figure 6—figure supplement 1E.
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Figure 7
Model of HUWE1-dependent TTP regulation.
(A) Model indicating the TTP regions in its C-terminus speculated to recruit PP1/2 and an unknown E3 ligase that ubiquitinates the zinc finger domain. (B) Model of TTP stability regulation through …
Tables
Table 1
Vectors.
| Plasmid | Purpose | Reference or source |
|---|---|---|
| pRRL-TRE3G-Cas9-P2A-GFP-PGK-IRES-rtTA3 | Dox inducible Cas9 | Johannes Zuber, IMP |
| pLX303-mCherry.TTP-P2A-BFP | TTP reporter | This study |
| pLX303-MYC-mCherry-P2A-3xHA.TTP | TTP reporter | This study |
| pLX303-MYC-mCherry-P2A-3xHA.TTP ΔC1 | TTP reporter | This study |
| pLX303-MYC-mCherry-P2A-3xHA.TTP ΔC2 | TTP reporter | This study |
| pLX303-MYC-mCherry-P2A-3xHA.TTP ΔC3 | TTP reporter | This study |
| pLX303-MYC-mCherry-P2A-3xHA.TTP ΔC4 | TTP reporter | This study |
| pLX303-MYC-mCherry-P2A-3xHA.TTP ΔC5 | TTP reporter | This study |
| pLX303-MYC-mCherry-P2A-3xHA.TTP ΔC6 | TTP reporter | This study |
| pLX303-MYC-mCherry-P2A-3xHA.TTP ΔN1 | TTP reporter | This study |
| pLX303-MYC-mCherry-P2A-3xHA.TTP ΔN2 | TTP reporter | This study |
| pLX303-MYC-mCherry-P2A-3xHA.TTP ΔN3 | TTP reporter | This study |
| pLX303-MYC-mCherry-P2A-3xHA.TTP ΔN4 | TTP reporter | This study |
| pLX303-MYC-mCherry-P2A-3xHA.TTP Δ259–278 | TTP reporter | This study |
| pLX303-MYC-mCherry-P2A-3xHA.TTP ΔC234-258 | TTP reporter | This study |
| pLX303-MYC-mCherry-P2A-3xHA.TTP Δ206–233 | TTP reporter | This study |
| pLX303-MYC-mCherry-P2A-3xHA.TTP KtoR | TTP reporter | This study |
| pLX303-MYC-mCherry-P2A-3xHA.TTP S52A S178A | TTP reporter | This study |
| pLX303-MYC-mCherry-P2A-3xHA.TTP ZNF C116R C139R | TTP reporter | This study |
| pLX303-MYC-mCherry-P2A-3xHA.TTP K97R | TTP reporter | This study |
| pLX303-MYC-mCherry-P2A-3xHA.TTP K115R | TTP reporter | This study |
| pLX303-MYC-mCherry-P2A-3xHA.TTP K133R | TTP reporter | This study |
| pLX303-MYC-mCherry-P2A-3xHA.TTP K135R | TTP reporter | This study |
| pLX303-MYC-mCherry-P2A-3xHA.TTP K141R | TTP reporter | This study |
| pLX303-MYC-mCherry-P2A-3xHA.TTP K97R/K115R | TTP reporter | This study |
| pLX303-MYC-mCherry-P2A-3xHA.TTP K97R/K115R/K133R | TTP reporter | This study |
| pLX303-MYC-mCherry-P2A-3xHA.TTP K97R/K115R/K133R/K135R | TTP reporter | This study |
| pLX303-MYC-mCherry-P2A-3xHA.TTP K135R/K141R | TTP reporter | This study |
| pLX303-MYC-mCherry-P2A-3xHA.TTP K133R/K135R/K141R | TTP reporter | This study |
| pLX303-MYC-mCherry-P2A-3xHA.TTP K115R/K133R/K135R/K141R | TTP reporter | This study |
| CMV-Flag-TTP | TTP reporter | Pavel Kovarik, Max Perutz Labs |
| DualCRISPR-hU6-sgRNA-mU6-sgRNA-EF1as-BFP | Dual sgRNA | de Almeida M, Hinterndorfer M et al, 2021 |
| pLentiv2-U6-PGK-iRFP670-P2A-Neo | Single sgRNA | de Almeida M, Hinterndorfer M et al, 2022 |
| pLentiv2-U6-PGK-BFP-P2A-Neo | Single sgRNA | de Almeida M, Hinterndorfer M et al, 2023 |
| PRRL-PBS-U6-sgRNA-EF1as-Thy1-P2A-NeoR (sgETN) | Library sgRNA | Johannes Zuber, IMP |
Table 2
sgRNA coding sequences.
| Gene | Species | Sequence (5' to 3') |
|---|---|---|
| ROSA_1 (RAW264.7 & BMDMs) | mouse | AGATGGGCGGGAGTCTTC |
| ROSA_2 (RAW264.7 & BMDMs) | mouse | TTTAGATGGGCGGGAGTCTTCGTTTA |
| Huwe1_1 (RAW264.7 & BMDMs) | mouse | GATTTGCTGCAGTTCCAAG |
| Huwe1_2 (RAW264.7 & BMDMs) | mouse | ATAAAATTCAAAGTGTAGTG |
| Psmb7_1 (RAW264.7 & BMDMs) | mouse | GCTGTAACAACTCTCGGG |
| Psmb7_2 (RAW264.7 & BMDMs) | mouse | GAAAACTGGCACTACCATCG |
| Vcpip1 (RAW264.7 & BMDMs) | mouse | GACGTGCTCTGGTTCGATG |
| Ppil4 (RAW264.7 & BMDMs) | mouse | GTGTTTGGTGAAGTGACAGA |
| Tceb1 (RAW264.7 & BMDMs) | mouse | GCTGAGAATGAAACCAACG |
| Ube3c (RAW264.7 & BMDMs) | mouse | GAGAGTCAAAGTTCAAAA |
| Ddx23 (RAW264.7 & BMDMs) | mouse | GGATGGAGCGGGAGACCAA |
| Cnot10 (RAW264.7 & BMDMs) | mouse | GATTTCACAGGGTAGCGG |
| Ttp (RAW264.7 & BMDMs) | mouse | GAAGCGGGCGTTGTCGCTACG |
| AAVS1_1 (RKO & HEK-293T) | human | CTGTGCCCCGATGCACAC |
| AAVS1_2 (RKO & HEK-293T) | human | GCTGTGCCCCGATGCACAC |
| HUWE1_1 (RKO & HEK-293T) | human | GTGCGAGTTATATCACTGGG |
| HUWE1_2 (RKO & HEK-293T) | human | GTGCGAGTTATATCACTGGGTGG |
| AAVS1_3 (A375 & SW620) | human | GCTGTGCCCCGATGCACAC |
| AAVS1_4 (A375 & SW620) | human | GCTTGGCAAACTCACTCTT |
| HUWE1_3 (A375 & SW620) | human | GTGCGAGTTATATCACTGGG |
| HUWE1_4 (A375 & SW620) | human | GACAGTGGAGAATATGTCA |
Table 3
Cells and culture conditions.
| Cell lines and primary cells | Type | Reference or source | Purpose | Media | Supplements |
|---|---|---|---|---|---|
| RAW264.7 | Murine macrophages | ATCC TIB-71 | parental cell line | Dulbecco’s modified Eagle’s medium (DMEM; Sigma-Aldrich, D6429) | 10% FBS (Sigma-Aldrich, F7524) and 1% penicillin/streptomycin (Sigma-Aldrich, P4333) |
| RAW264.7-Dox-Cas9 | Murine macrophages | This study | Dox-inducible Cas9 | Dulbecco’s modified Eagle’s medium (DMEM; Sigma-Aldrich, D6429) | 10% FBS (Sigma-Aldrich, F7524) and 1% penicillin/streptomycin (Sigma-Aldrich, P4333) |
| RAW264.7-Dox-Cas9 mCherry-TTP-P2A-BFP | Murine macrophages | This study | mCherry-TTP reporter | Dulbecco’s modified Eagle’s medium (DMEM; Sigma-Aldrich, D6429) | 10% FBS (Sigma-Aldrich, F7524) and 1% penicillin/streptomycin (Sigma-Aldrich, P4333) |
| RAW264.7-Dox-Cas9 mCherry-IkBα-P2A-BFP | Murine macrophages | This study | mCherry-IkBα reporter | Dulbecco’s modified Eagle’s medium (DMEM; Sigma-Aldrich, D6429) | 10% FBS (Sigma-Aldrich, F7524) and 1% penicillin/streptomycin (Sigma-Aldrich, P4333) |
| RAW264.7-Dox-Cas9 3xHA-TTP | Murine macrophages | This study | 3xHA-TTP reporter | Dulbecco’s modified Eagle’s medium (DMEM; Sigma-Aldrich, D6429) | 10% FBS (Sigma-Aldrich, F7524) and 1% penicillin/streptomycin (Sigma-Aldrich, P4333) |
| Bone Marrow Derived Macrophages, BMDMs | Murine macrophages | This study | constitutive Cas9 expression | Dulbecco’s modified Eagle’s medium (DMEM; Sigma-Aldrich, D6429) | 10% FBS (Sigma-Aldrich, F7524) and 1% penicillin/streptomycin (Sigma-Aldrich, P4333) |
| HEK293T | Human kidney neural tissue | CRL-3216 | 3xHA-TTP mutants | Dulbecco’s modified Eagle’s medium (DMEM; Sigma-Aldrich, D6429) | 10% FBS (Sigma-Aldrich, F7524) and 1% penicillin/streptomycin (Sigma-Aldrich, P4333) |
| Lenti-X 293T | Human kidney neural tissue | Takara, Cat# 632180 | VLP production | Dulbecco’s modified Eagle’s medium (DMEM; Sigma-Aldrich, D6429) | 10% FBS (Sigma-Aldrich, F7524) and 1% penicillin/streptomycin (Sigma-Aldrich, P4333) |
| RKO | human colon carcinoma | de Almeida M, Hinterndorfer M et al, 2021 | Dox-inducible Cas9 | RPMI 1640 (Thermo Fisher Scientific, 21875) | 10% FBS (Sigma-Aldrich, F7524), L-glutamine (4 mM, Gibco), sodium pyruvate (1 mM, Sigma-Aldrich), and 1% penicillin/streptomycin (Sigma-Aldrich, P4333) |
| A375 | human melanoma | This study | Dox-inducible Cas9 | Dulbecco’s modified Eagle’s medium (DMEM; Sigma-Aldrich, D6429) | 10% FBS (Sigma-Aldrich, F7524), L-glutamine (4 mM, Gibco) and 1% penicillin/streptomycin (Sigma-Aldrich, P4333) |
| SW620 | human colon carcinoma | This study | Dox-inducible Cas9 | Dulbecco’s modified Eagle’s medium (DMEM; Sigma-Aldrich, D6429) | 10% FBS (Sigma-Aldrich, F7524), L-glutamine (4 mM, Gibco) and 1% penicillin/streptomycin (Sigma-Aldrich, P4333) |
Table 4
reagents.
| Description | Abbreviation | Application | Dilution/concentration | Manufacturer | Catalogue number |
|---|---|---|---|---|---|
| Lipopolysaccharides from Escherichia coli O55:B5 | LPS | Cell culture | 10 ng/ml | Sigma-Aldrich | L2637 |
| Cycloheximide | CHX | Cell culture | 40 μg/m | Sigma-Aldrich | C1988 |
| MG132 | MG132 | Cell culture | 10 μM | Sigma-Aldrich | M7449 |
| Epoxomicin | EPX | Cell culture | 10 μM | Gentaur Molecular Products | 607-A2606 |
| TAK-243 | Cell culture | 0.5 μM | ChemScence | CS-0019384 | |
| Doxycycline hyclate | DOX | Cell culture | 500 ng/ml | Sigma-Aldrich | D9891 |
| G418 disulfate salt | G418 | Cell culture | 0.5–1 mg/ml | Sigma-Aldrich | A1720 |
| PH-797804, p38 inhibitor | p38i | Cell culture | 1 μM | Selleckchem | S2726 |
| JNK Inhibitor II, JNK inhibitor | JNKi | Cell culture | 20 μM | Sigma-Aldrich | 420119 |
| PF-3644022, MK2 inhibitor | MK2i | Cell culture | 10 μM | Sigma-Aldrich | PZ0188 |
| U0126, MEKi inhibitor | ERKi | Cell culture | 250 nM | Cell Signaling Technology | 9903 |
| Okadaic Acid | OA | Cell culture | 1 μM | Cell Signaling Technology | 5934 |
| Calyculin A | CalycA | Cell culture | 50 nM | Cell Signaling Technology | 9902 |
| Etoposide | Cell culture | 5 μM | Sigma-Aldrich | E1383 | |
| Brefeldin A | Cell culture | 10 ug/ml | Sigma-Aldrich | B7651 |
Table 5
antibodies.
| Target | Application | Dilution | Conjugate | Manufacturer | Catalogue number | Name | Type |
|---|---|---|---|---|---|---|---|
| TTP | Western blot | 1:1000 | Cell Signaling Technology | 71632 | D1I3T | Primary | |
| Myc Tag | Western blot | 1:5000 | Sigma-Aldrich | 05–724 | 4A6 | Primary | |
| HA tag | Western blot | 1:1000 | Cell Signaling Technology | 3724 | C29F4 | Primary | |
| HECTH9 | Western blot | 1:1000 | Cell Signaling Technology | 5695 | AX8D1 | Primary | |
| Lasu1/Ureb1 | Western blot | 1:1000 | Bethyl | A300-486A | Primary | ||
| Vinculin | Western blot | 1:1000 | Sigma-Aldrich | V9131 | V9131 | Primary | |
| phospho-p38 MAPK, Thr180/Tyr182 | Western blot | 1:1000 | Cell Signaling Technology | 9211 | Primary | ||
| p38 MAPK | Western blot | 1:1000 | Cell Signaling Technology | 9212 | Primary | ||
| phospho-SAPK/JNK, Thr183/Tyr185 | Western blot | 1:1000 | Cell Signaling Technology | 9251 | Primary | ||
| SAPK/JNK | Western blot | 1:1000 | Cell Signaling Technology | 9252 | Primary | ||
| phospho-p44/42 MAPK (Erk1/2), Thr202/Tyr204 | Western blot | 1:1000 | Cell Signaling Technology | 9101 | Primary | ||
| p44/42 MAPK (Erk1/2) | Western blot | 1:1000 | Cell Signaling Technology | 4695 | 137F5 | Primary | |
| p-MK2 (Thr334) | Western blot | 1:1000 | Cell Signaling Technology | 3007 | 27B7 | Primary | |
| MK2 | Western blot | 1:1000 | Cell Signaling Technology | 3042 | Primary | ||
| p-p53, Ser15 | Western blot | 1:1000 | Cell Signaling Technology | 9284 | Primary | ||
| p-p53 | Western blot | 1:1000 | Cell Signaling Technology | 2524 | 1C12 | Primary | |
| ZFP36L1/2 | Western blot | 1:1000 | Proteintech | 12306–1-AP | 12306–1-AP | Primary | |
| Ubiquitin | Western blot | 1:1000 | Santa Cruz Biotechnology | sc-8017 | P4D1 | Primary | |
| HRP-β-actin | Western blot | 1:20000 | HRP | Abcam | ab49900 | AC-15 | Primary |
| HRP anti-rabbit IgG | Western blot | 1:3500 | HRP | Cell Signaling Technology | 7074 | Secondary | |
| HRP anti-mouse IgG | Western blot | 1:3500 | HRP | Cell Signaling Technology | 7076 | Secondary | |
| TTP | FACS | 1:100 | Cell Signaling Technology | 71632 | D1I3T | Primary | |
| HECTH9 | FACS | 1:100 | Cell Signaling Technology | 5695 | AX8D1 | Primary | |
| TNF alpha | FACS | 1:100 | APC | eBioscience | 17-7321-82 | MP6-XT22 | Primary |
| Rat IgG1 kappa Isotype Control | FACS | 1:500 | APC | eBioscience | 17-4301-82 | eBRG1 | Primary |
| APC anti-CD90.1/Thy1.1 | FACS | 1:500 | APC | BioLegend | 202526 | Secondary | |
| Alexa Fluor Plus 594 anti-Mouse IgG | FACS | 1:500 | Alexa Fluor 594 | Thermo Fisher Scientific | A-21201 | Secondary | |
| Alexa Fluor Plus 680 anti-Rabbit IgG | FACS | 1:500 | Alexa Fluor 680 | Thermo Fisher Scientific | A-21076 | Secondary | |
| APC anti-F4/80 | FACS | 1:100 | APC | Thermo Fisher Scientific | 17-4801-82 | BM8 | Secondary |
| TruStain FcX mouse Fc Receptor CD16/32 | FACS | 1:100 | BioLegend | 101319 | |||
| IgG Isotype Control | IP | 1:300 | Cell Signaling Technology | 2729 | Primary | ||
| TTP | IP | 1:100 | Cell Signaling Technology | 71632 | D1I3T | Primary | |
| HA tag | IP | 1:100 | Cell Signaling Technology | 3724 | C29F4 | Primary |
Table 6
qPCR primers.
| Target | Primer | Sequence (5' to 3') |
|---|---|---|
| Il6 | FWD | CCAGAAACCGCTATGAAGTTCC |
| REV | TTGTCACCAGCATCAGTCCC | |
| Zfp36 | FWD | CTCTGCCATCTACGAGAGCC |
| REV | GATGGAGTCCGAGTTTATGTTCC | |
| Tnf | FWD | GATCGGTCCCCAAAGGGATG |
| REV | CACTTGGTGGTTTGCTACGAC | |
| pre-Tnf | FWD | GGCAAAGAGGAACTGTAAG |
| REV | CCATAGAACTGATGAGAGG | |
| Gapdh | FWD | ATGGTGAAGGTCGGTGTGA |
| REV | TGAAGGGGTCGTTGATGG |
Table 7
NGS library primers.
| PCR 1 | |||
|---|---|---|---|
| Primer_name | Direction | Sequence | Comments |
| sgDeepSeq_rev_XXXX | Rv | CTCTTTCCCTACACGACGCTCTTCCGATCT NNNNNNCTCATTCCAGCATAGCTCTTAAAC | Library preparation 1st PCR |
| sgDeepSeq_rev_XXXX | Rv | CTCTTTCCCTACACGACGCTCTTCCGATCT NNNNNNTCGATTCCAGCATAGCTCTTAAAC | Library preparation 1st PCR |
| sgDeepSeq_rev_XXXX | Rv | CTCTTTCCCTACACGACGCTCTTCCGATCT NNNNNNCCTATTCCAGCATAGCTCTTAAAC | Library preparation 1st PCR |
| sgDeepSeq_rev_XXXX | Rv | CTCTTTCCCTACACGACGCTCTTCCGATCT NNNNNNGAACTTCCAGCATAGCTCTTAAAC | Library preparation 1st PCR |
| sgDeepSeq_rev_XXXX | Rv | CTCTTTCCCTACACGACGCTCTTCCGATCT NNNNNNATCCTTCCAGCATAGCTCTTAAAC | Library preparation 1st PCR |
| sgDeepSeq_rev_XXXX | Rv | CTCTTTCCCTACACGACGCTCTTCCGATCT NNNNNNACTCTTCCAGCATAGCTCTTAAAC | Library preparation 1st PCR |
| sgDeepSeq_rev_XXXX | Rv | CTCTTTCCCTACACGACGCTCTTCCGATCT NNNNNNCTTCTTCCAGCATAGCTCTTAAAC | Library preparation 1st PCR |
| sgDeepSeq_rev_XXXX | Rv | CTCTTTCCCTACACGACGCTCTTCCGATCT NNNNNNCAAGTTCCAGCATAGCTCTTAAAC | Library preparation 1st PCR |
| sgDeepSeq_rev_XXXX | Rv | CTCTTTCCCTACACGACGCTCTTCCGATCT NNNNNNTGAGTTCCAGCATAGCTCTTAAAC | Library preparation 1st PCR |
| sgDeepSeq_rev_XXXX | Rv | CTCTTTCCCTACACGACGCTCTTCCGATCT NNNNNNTTCGTTCCAGCATAGCTCTTAAAC | Library preparation 1st PCR |
| sgDeepSeq_rev_XXXX | Rv | CTCTTTCCCTACACGACGCTCTTCCGATCT NNNNNNTAGGTTCCAGCATAGCTCTTAAAC | Library preparation 1st PCR |
| sgDeepSeq_rev_XXXX | Rv | CTCTTTCCCTACACGACGCTCTTCCGATCT NNNNNNTCTGTTCCAGCATAGCTCTTAAAC | Library preparation 1st PCR |
| Fwd1_hybrid_P7_Nras | Fwd | GCATACGAGATAGCTAGCCACC | Library preparation 1st PCR |
| PCR 2 | |||
| Primer_name | Direction | Sequence | Comments |
| Rev2_p5_sgDeepSeq | Rv | AATGATACGGCGACCACCGAGATCTACACT CTTTCCCTACACGACGCT | Library preparation 2nd PCR |
| Fwd2_p7_sgDeepSeq | Fwd | CAAGCAGAAGACGGCATACGAGATAGCTAGCCACC | Library preparation 2nd PCR |
