EMBO/SourceData huggingface dataset:
Figure legend: "U2OS cells were labeled with CldU for 10 min, followed by increasing IdU labelling times and subjected to DNA stretching. IdU track length after 10-80 min. Theoretical IdU track lengths are shown in red; the theoretical 10-min points were considered equal to the experimental values obtained for siLuc or siChk1, and the following theoretical points were calculated proportionally. Data were calculated as the mean (+SD) of mean IdU track lengths (B) n=4 for IdU times 20-60 min or n=3 for IdU times 10 and 80 min; for each experiment and condition 60-100 fibers were scored."
NCBI gene IDs:
"Luc: 
Chk1: 1111"
Figure legend: "DNA combing from HCT116 cells labeled with CldU and IdU for 20 min. The upper left panel shows the labelling scheme. The upper right panel shows representative sister replication forks that emanate from the same origin (ORI=origin of replication); a pair of sister forks is considered asymmetric if their lengths (CldU+IdU) differ in more than 33%. The percentage of asymmetric forks (lower left panel; a Western Blot of Chk1 and Ku80, loading control, performed in parallel to the combing experiments, is shown) and the lengths of each pair of forks (lower right panel; points outside of the dotted lines correspond to asymmetric forks) are shown. Data (siLuc, n=131; siChk1, n=117) come from 2 independent experiments; the numbers indicate the mean (+SD) percentage of asymmetric forks."
NCBI gene IDs:
"Luc: 
Chk1: 1111"
Figure legend: "Representative images of siChk1-depleted U2OS cells showing nuclear localization of GFP-Polη-WT/S687A/S687D. Numbers indicate percentage of cells showing nuclear GFP-Polη (mean+SD, n=2). Scale bar: 100 μm."
NCBI gene IDs:
"GFP: 
Polη: 
Chk1: 1111"
Figure legend: "Western Blot of GFP-Polη in U2OS cells, 48/24 h after transfection with siRNA/GFP(-Polη). Actin was used as loading control."
NCBI gene IDs:
"GFP: 
Polη: "
Figure legend: "IdU track lengths from U2OS cells transfected with GFP (-) or GFP-Polη-WT/S687A/S687D. &gt;180 fibers obtained from 2 independent experiments were measured for each condition."
NCBI gene IDs:
"GFP: 
Polη: "
Figure legend: "Number of foci per cell after CSK extraction in siLuc and siChk1-depleted U2OS cells transfected with GFP-Polη-WT/S687A/S687D. &gt;150 cells/sample were analyzed in 4 independent experiments. p&lt;0.05 was considered significant."
NCBI gene IDs:
"GFP: 
Luc: 
Chk1: 1111
Polη: 353497"
Figure legend: "IdU track lengths and percentage of origin firing (mean+SD) from U2OS cells transfected or not with siCDT1. Track lengths from &gt;290 fibers obtained from 3 independent experiments were measured for each condition. To calculate origin frequency &gt;700 DNA fibers from 3 independent experiments were scored."
NCBI gene IDs:
"CDT1: 81620"
Figure legend: "IdU track lengths and percentage of origin firing (mean+SD) from U2OS cells transfected or not with GFP-Polη. &gt;180 (track length) and &gt;400 (origin frequency) DNA fibers obtained from 2 independent experiments were measured for each condition."
NCBI gene IDs:
"GFP: 
Polη: "
Figure legend: "DSB accumulation measured by neutral comet assay after 8 h treatment with Chk1i, in the presence of 5 μM NS, siCDC7 or 10 μM roscovitine. 85-170 cells/sample were analyzed in 2 independent experiments. Representative images of data shown in (D). Scale bar: 25 μm."
NCBI gene IDs:
"CDC7: 8317"
Figure legend: "Sensitivity of U2OS cells to Chk1 inhibition, roscovitine (5 μM) and Polη depletion. Cell number was determined 4 days after a 24 h treatment (5 days in total) with Chk1i±Rosc. Data represent the mean (+SD) of 3 independent experiments."
NCBI gene IDs:
"Polη: 353497"
Figure legend: "Sensitivity of U2OS cells transduced with shRNA targeting Chk1 (shChk1) or non-targeting shRNA (shScr) and Lenti-GFP-Polη or Lenti-GFP (-). Cell number was determined 7 days after transduction (5 days after maximal downregulation). Data represent the mean (+SD) of 3 independent experiments."
NCBI gene IDs:
"GFP: 
Polη: 
Chk1: 1111"
Figure legend: "IdU track lengths from U2OS cells. &gt;170 DNA fibers obtained from 2 independent experiments were measured for each condition. For comparison, the data showing that Polη is required for roscovitine-dependent rescue of fork elongation in Chk1-depleted cells (Fig 3B) is shown on the right. The lower panel shows the corresponding Western Blot of Chk1, Polη and CDC45. Actin was used as a loading control."
NCBI gene IDs:
"Chk1: 1111
Polη: 353497"
Figure legend: "Sensitivity of U2OS cells to Chk1 and CDC45 depletion. Cell number was determined 7 days after transfection (5 days after maximal downregulation). Data represent the mean (+SD) of 2 independent experiments. Representative image of data shown in (E). Scale bar: 100 μm."
NCBI gene IDs:
"CDC45: 8318
Chk1: 1111"
Figure legend: "Distribution of the plasma membrane reporter PH::GFP in control cells (A-A'') and in cells where dynamin activity had been blocked using a temperature-sensitive allele of dynamin (shibirets In (B), was inactivated at the onset of tube formation (C-D) Distribution of PH::GFP fluorescence intensity in control and shibirets cells. Data from 1-2 minute-interval time lapses was collected in windows of 20 minutes each (except for t=0). (C) Proportion of signal in the apical and in the basal membrane compartment over time in control cells (n=4) and also in cells where dynamin was inactivated (n=5), *p=0.015, Mann-Whitney test. (D) Total fluorescence intensity of PH::GFP over time in controls (n=4) and in cells where dynamin was inactivated (n=5), n.s.: p=0.063, Mann-Whitney test."
NCBI gene IDs:
"shibire: 45928"
Figure legend: " Distribution of the plasma membrane reporter PH::GFP in cells where dynamin activity had been blocked using a temperature-sensitive allele of dynamin (shibirets in (E) it was inactivated ~15 minutes after the tube had begun to form. "
NCBI gene IDs:
"shibire: 45928"
Figure legend: " (A-F) TEM tomograms and 3D reconstructions of terminal cells in shibirets embryos. (A) Control. (B-F) Kept at 34ºC for 1 (B, E) or for 2 hours (C, D, F). Boxed regions in (A) and (B) are magnified in (A'') and (B''). (A', B') Reconstructed 3D models. The tube membrane (cyan) is characterized by the electron-dense apical extracellular matrix (aECM). Invaginations with connections that can be traced to the tube membrane are red, invaginations from the basal plasma membrane are green. The basal membrane is light pink. (C-D') shibirets terminal cell after 2 hours at 34ºC. (C') 3D reconstruction, color coding for basal and tube membrane is the same as above. Membrane that is continuous with both the apical and the basal membrane is shown in green, purple and dark blue. (D-D') higher magnification of a region in the same cell at a level where membrane sheets (purple, dark blue) bridge the apical and basal plasma membrane domains. (E-E'') Two focal planes and model of a tomogram from the cell shown in (B) where a basal (pink arrowhead) and an apical invagination (blue) are seen in close proximity (closest distance is marked by cyan and blue arrowheads). (F-F') apical membrane overgrowth regions of the cell shown in (C). The genotype of the embryos was shibirets; btl&gt;KDEL::RFP, Par3::YFP. The cell shown in (C-D) was found and acquired without the CLEM approach. "
NCBI gene IDs:
"KDEL: 
Par3: 
btl: 39564
shibire: 45928"
Figure legend: "(A-B) Terminal cells expressing the membrane marker CD4::mIFP under btl-gal4 and FGFR::GFP under its own promoter (from the fTRG library). (A) Time lapse imaging of a control cell. Blue arrowheads point to filopodia and basal plasma membrane and red ones point to puncta containing CD4::mIFP and FGFR::GFP. (B) shibirets cell imaged before dynamin inactivation, after 23 and 45 minutes of inactivation, and after 30 minutes of recovery. White arrowheads point to FGFR::GFP accumulation at the apical compartment."
NCBI gene IDs:
"gal4: 
btl: 39564
shibire: 45928"
Figure legend: "High time resolution imaging of the membrane marker CD4::mIFP expressed under btl-gal4. (A) Example of a large CD4 vesicle (arrowhead) rapidly moving towards the tip of the terminal cell. (B-D) Analysis of 28 CD4 vesicles in five terminal cells."
NCBI gene IDs:
"gal4: 
btl: 39564"
Figure legend: "(A-F) Terminal cells expressing CD4::mIFP and FYVE::GFP under btl-gal4. The outline of the cells was traced using the CD4::mIFP signal and is shown as a blue dashed line. (A-C) Control cell. (D-F) shibirets cell before 34ºC (D), after 30 minutes at 34ºC (E) and after 30 minutes of recovery (F). Red arrowheads: FYVE::GFP puncta."
NCBI gene IDs:
"gal4: 
btl: 39564
shibire: 45928"
Figure legend: " (G-H) Number of FYVE::GFP vesicles in 4 control cells (G) and in 6 shibirets cells (H). Box plots represent median, interquartile range (IQR), and IQR*1.5 below and above the IQR. Significance in (H) was assessed using one-way ANOVA with Geisser-Greenhouse correction for paired data and Dunnett's multiple comparisons test. *p=0.0288, ***p=0.0016. "
NCBI gene IDs:
"shibire: 45928"
Figure legend: "(I) shrbO3/G5 mutant cell expressing CD4::mIFP under btl-gal4. Arrowhead: CD4::mIFP aggregation."
NCBI gene IDs:
"gal4: 
btl: 39564
shrb: 35933"
Figure legend: "(J-K) Terminal cells over-expressing Shrb::GFP under btl-gal4 together with PH::mCherry (J), or together with Spin::RFP and CD4::mIFP (K). Arrowheads: Shrb::GFP accumulations."
NCBI gene IDs:
"gal4: 
GFP: 
btl: 39564
Shrb: 35933"
Figure legend: "(L-M) Dorsal trunk cells stained for Serp. In control embryos (L), Serp is seen exclusively in the tracheal lumen. The cells themselves are not visible in these images. In embryos expressing Shrb::GFP under btl-gal4 (M), Serp is also seen inside the cells, usually in association with or surrounded by Shrb::GFP. Boxed regions are magnified in (L'-M') and shown as single confocal planes."
NCBI gene IDs:
"gal4: 
GFP: 
btl: 39564
Shrb: 35933"
Figure legend: "C. Cathepsin activity  assay in WT and Tpl2[D270A] BMDMs 0.5 h after uptake of  latex beads. BMDMs were stained with the Magic Red cathepsin L substrate (red). Average fluorescence intensity of the cathepsin probe per cell was quantified (n = 40-51 cells). E. pH assay in WT and Tpl2[D270A] BMDMs upon 0.5 h after incubation with  latex beads. BMDMs were stained  with the LysoTracker Red DND-99 dye  (red). Average  fluorescence intensity of the  Lysotracker Red probe per cell was quantified (n = 95-126 cells). Data information: One representative experiment out of three shown. Error bars and shaded areas represent SEM. **** P &lt; 0.0001. Student's unpaired t-test."
NCBI gene IDs:
"Tpl2: 26410"
Figure legend: "G. Reactive oxygen species assay in WT and Tpl2[D270A] BMDMs upon 0.5 h after incubation with latex beads. BMDMs were stained with the ROS Deep Red dye. Average fluorescence intensity of the ROS Deep Red probe per cell was quantified (n = 80-110 cells) Data information: One representative experiment out of three shown. Error bars and shaded areas represent SEM. **** P &lt; 0.0001. Student's unpaired t-test."
NCBI gene IDs:
"Tpl2: 26410"
Figure legend: "Experiments were performed using murine BMDMs. (C) Cell extracts from LPS-stimulated Nfkb1[SSAA] and Nfkb1[SSAA]/Tpl2[D270A] BMDMs were immunoblotted  for the indicated antigens."
NCBI gene IDs:
"Tpl2: 26410
Nfkb1: 18033"
Figure legend: "C. Protein intensities of V-ATPase subunits from phagosomes purified from WT and Tpl2[D270A] BMDMs, (n = 3 biological replicates). Data information: Data were analysed by Student's t-test. Error bars represent SEM. * P &lt; 0.05, ** P &lt; 0.01, *** P &lt; 0.001, **** P &lt; 0.0001."
NCBI gene IDs:
"Tpl2: 26410"
Figure legend: "D. Protein intensities of RAB5 and LAMP-1 from phagosome proteome analysis of WT and Tpl2[D270A] BMDMs (n = 3 biological replicates) (left). Immunoblot of isolated phagosomes from WT and Tpl2[D270A] BMDMs probed for RAB5,  LAMP-1  and vimentin. Phagosomal fractions of two biological replicates were pooled. One representative experiment out of two shown (right) (n = 2). Data information: Data were analysed by Student's t-test. Error bars represent SEM. * P &lt; 0.05, ** P &lt; 0.01, *** P &lt; 0.001, **** P &lt; 0.0001. "
NCBI gene IDs:
"Tpl2: 26410"
Figure legend: "A. TPL-2-dependent phosphoproteome following phagocytosis of latex beads  (0.5 h) was determined by TMT mass spectrometry. Volcano plot representing the significance (-log10 P-values after Welch's t-test) vs. phosphorylation  fold change (Welch difference ratios) between WT and Tpl2[D270A] BMDMs. Five biological replicates were analysed per genotype (n = 5). Three of the most highly and significantly downregulated phospho-sites in Tpl2[D270A]  BMDMs relative to WT, as well as unaltered DMXL1 phospho-sites, are shown."
NCBI gene IDs:
"Tpl2: 26410"
Figure legend: "U-2 OS cells stably expressing GFP-MDC1 or GFP-53BP1 were treated with 1 Gy of IR and analyzed by live cell microscopy at 15 min intervals. Representative images are provided and kinetics of GFP-MDC1 and GFP-53BP1 foci formation and dissolution are shown as single cell tracks. Bold lines represent averages from n=29 for GFP-MDC1 and n=18 for GFP-53BP1 cells."
NCBI gene IDs:
"GFP: 
MDC1: 9656
53BP1: 27223"
Figure legend: "GFP-53BP1 cells were treated with 25 ng/ml NCS to induce DNA breaks and imaged at 30 minutes intervals. Examples of GFP-53BP1 fusions (green arrowheads and magnified regions) and fissions (blue arrowheads and magnified regions) are shown."
NCBI gene IDs:
"GFP: 
53BP1: 27223"
Figure legend: "53BP1-RFP cells, in which the endogenous 53BP1 gene locus had been engineered by CRISPR/Cas9 to express 53BP1-mScarlet from the natural promoter, were treated with NCS (25 ng/ml) and imaged at 30 minutes intervals. Examples of 53BP1 fusions (green arrowheads and magnified regions) and fissions (blue arrowheads and magnified regions) are shown on the left and in higher magnification on the right."
NCBI gene IDs:
"CRISPR: 
RFP: 
53BP1: 7158"
Figure legend: "GFP-53BP1 cells were left untreated, or treated with APH (0.5 μM) or ATRi (1 μM) to induce replication stress-associated heritable DNA lesions, and cells were imaged at 30 minutes intervals. Examples of 53BP1 fusions (green arrowheads and magnified regions) are shown."
NCBI gene IDs:
"GFP: 
53BP1: 27223"
Figure legend: "Light-induced optoDroplet formation of Cry2-mCherry-53BP1, Cry2-mCherry-53BP1 W1495A, and Cry2-mCherry-MDC1. Cells were imaged at 15 seconds intervals. Representative images of optoDroplet formation before and 6 minutes after light induction are shown. Quantifications from single-cell QIBC analysis of 2-3 independent experiments are shown with mean (solid line) and standard deviation from the mean (dashed lines) indicated."
NCBI gene IDs:
"mCherry: 
Cry2: 839529
MDC1: 9656
53BP1: 7158"
Figure legend: "Cry2-mCherry-53BP1 W1495A optoDroplet fusion at 15 seconds time resolution. Fusing optodroplets are highlighted by green arrowheads and in the magnified insets."
NCBI gene IDs:
"mCherry: 
Cry2: 839529
53BP1: 7158"
Figure legend: "Light-induced optoDroplet formation of the indicated Cry2-mCherry-53BP1 constructs. Cells were imaged at 15 seconds intervals. Representative images and quantifications of optoDroplet formation before and 6 minutes after light induction are shown. Quantifications from single-cell QIBC analysis of 2-3 independent experiments are shown with mean (solid line) and standard deviation from the mean (dashed lines) indicated. Red bars indicate the part of 53BP1 that was expressed as Cry2-mCherry fusion."
NCBI gene IDs:
"mCherry: 
Cry2: 839529
53BP1: 7158"
Figure legend: "Cry2-mCherry-53BP1 1203-1972 W1495A optoDroplets undergo fusion events upon light induction. Fusing optodroplets are highlighted by green arrowheads and in the magnified insets."
NCBI gene IDs:
"mCherry: 
Cry2: 839529
53BP1: 7158"
Figure legend: "Concentration-dependent optoDroplet formation exemplified by two neighboring cells that differ in their Cry2-mCherry-53BP1 1203-1972 W1495A expression level. Cells were imaged at 15 seconds intervals. Quantifications of optoDroplet formation in lowly expressing cells (mClow) and highly expressing cells (mChigh) from single-cell QIBC analysis of 3 independent experiments are shown with mean (solid line) and standard deviation from the mean (dashed lines) indicated."
NCBI gene IDs:
"mCherry: 
Cry2: 839529
53BP1: 7158"
Figure legend: "Cry2-mEGFP-53BP1 1203-1972 W1495A assembles into FokI-induced DNA repair compartments within seconds after light induction. FokI was induced for 1h where indicated before cells were light-activated and imaged at 15 seconds intervals. Red arrowheads mark the FokI-induced lesion with mCherry accumulation."
NCBI gene IDs:
"mEGFP: 
Cry2: 839529
53BP1: 7158"
Figure legend: "Cry2-mCherry-53BP1 1203-1972 W1495A optoDroplets were induced as indicated and fixed 5 minutes later. Endogenous BRCA1 and p53 were co-stained."
NCBI gene IDs:
"mCherry: 
Cry2: 839529
53BP1: 7158"
Figure legend: "As in (A), comparing Cry2-mCherry-53BP1 constructs with and without the BRCT domains. Quantifications of p53 assembly into 53BP1 optoDroplets from single-cell QIBC analysis of 500-600 cells per condition are shown below with mean (solid line) and standard deviation from the mean (dashed lines) indicated. Red bars indicate the part of 53BP1 that was expressed as Cry2-mCherry fusion."
NCBI gene IDs:
"mCherry: 
Cry2: 839529
53BP1: 7158"
Figure legend: "qPCR of p21 induction upon DNA damage (10 Gy, 2h) with and without 0.4 M sorbitol. Mean ± standard deviation is indicated."
NCBI gene IDs:
"p21: 1026"
Figure legend: "A-B HT‑29 cells were transfected with non-silencing control siRNA (sictr) or siRNAs against USP22 (siUSP22) for 48 h at 20 nM. After transfection, cells were treated with 20 µM zVAD.fmk, 0.5 µM BV6 and 1 ng/ml TNFα either for 6 h and analyzed by Western blotting (A) or for 18 h, and the percentage of PI-positive cells was assessed by fluorescence-based PI staining (B). Vinculin served as a loading control."
NCBI gene IDs:
"USP22: 23326"
Figure legend: "C HT‑29 CRISPR/Cas9 control (ctr) and USP22 KO cells were analyzed by Western blotting for USP22 expression. GAPDH served as loading control."
NCBI gene IDs:
"Cas9: 
CRISPR: 
USP22: 23326"
Figure legend: "D HT‑29 control and USP22 KO cells were treated with 20 µM zVAD.fmk, 0.5 µM BV6 and 1 ng/ml TNFα after pre-incubation with 30 µM Nec1-s and 20 µM GSK'872 for 1 h and incubated for 18 h before fluorescence-based quantification of PI-positive cells."
NCBI gene IDs:
"USP22: 23326"
Figure legend: "E HT‑29 control and USP22 KO cells, generated with 2 guide RNAs (2g), expressing empty vector (EV) or PAM mutated 3xFLAG-HA-USP22 WT (USP22 PAM), C185S (USP22 PAM C185S) or C185A (USP22 PAM C185A) were analyzed by Western blotting for USP22 expression levels. β-Actin was used as a loading control."
NCBI gene IDs:
"FLAG: 
HA: 
USP22: 23326"
Figure legend: "F HT-29 control and USP22 KO cells, generated with 2 guide RNAs (2g), expressing empty vector (EV) or PAM mutated 3xFLAG-HA-USP22 WT (USP22 PAM), C185S (USP22 PAM C185S) or C185A (USP22 PAM C185A) were stimulated with 20 µM zVAD.fmk, 0.5 µM BV6, 1 ng/ml TNFα for 18 h. The percentage of PI-positive cells was assessed by fluorescence-based PI staining."
NCBI gene IDs:
"FLAG: 
HA: 
USP22: 23326"
Figure legend: "A HT‑29 control and USP22 KO cells were stimulated with 20 µM zVAD.fmk, 0.5 µM BV6 and 1 ng/ml TNFα for the indicated time points. Detection of indicated proteins was carried out by Western blotting. GAPDH served as a loading control."
NCBI gene IDs:
"USP22: 23326"
Figure legend: "B HT‑29 control and USP22 KO cells were stimulated with 20 µM zVAD.fmk, 0.5 µM BV6 and 1 ng/ml TNFα for 4 h. Detection of indicated proteins was carried out by Western blotting. β-Actin served as a loading control."
NCBI gene IDs:
"USP22: 23326"
Figure legend: "C HT‑29 control and USP22 KO cells were incubated with 30 µM Nec1-s or 20 µM Dab for 18 h, as indicated. Cell were stimulated with 20 µM zVAD.fmk, 0.5 µM BV6 and 1 ng/ml TNFα for 5 h. 100 μg of each lysate were incubated with 400 U/μl λ-phosphatase for 30 min at 30 °C. Protein expression of RIPK3 was monitored by Western blotting. β-Actin was used as loading control. High molecular weight RIPK3 'smears' were quantified after λ-phosphatase treatment and normalized to total RIPK3 and β-Actin levels."
NCBI gene IDs:
"USP22: 23326"
Figure legend: "A HeLa TRex RIPK3 CRISPR/Cas9 control (ctr) and USP22 KO cells were treated with 1 µg/ml Dox overnight. Protein expression of induced Strep-RIPK3 was analyzed by Western blotting. GAPDH served as loading control. The asterisk marks an unspecific band."
NCBI gene IDs:
"Cas9: 
CRISPR: 
USP22: 23326"
Figure legend: "B HeLa TRex RIPK3 control and USP22 KO cells were incubated with 1 µg/ml Dox for 18 h before pre-treatment with 20 µM zVAD.fmk, 5 µM BV6 for 1 h. After pre-treatment, 10 ng/ml TNFα was added and cell death was measured after 4 and 5 h by analysis of PI-positive nuclei."
NCBI gene IDs:
"USP22: 23326"
Figure legend: "C HeLa TRex RIPK3 control and USP22 KO cells were pre-treated with 20 µM zVAD.fmk, 5 µM BV6 for 1 h. After pre-treatment, 10 ng/ml TNFα were added for 1, 2, 3, 4 and 5 h. Protein expression of phosphorylated RIPK1, total RIPK1, total RIPK3, phosphorylated MLKL, total MLKL and USP22, without (left) or with (right) 1 µg/ml Dox treatment overnight, was monitored by Western blotting. GAPDH was used as a loading control."
NCBI gene IDs:
"USP22: 23326"
Figure legend: "D HT-29 control cells and RIPK3 KO cells re-expressing PAM-mutated Dox-inducible RIPK3 WT were incubated overnight with 1 µg/ml Dox. Cells were pre-treated with 20 µM zVAD.fmk, 5 µM BV6 for 1 h. After pre-treatment, 10 ng/ml TNFα were added for 2 h, as indicated. Strep-RIPK3 was immunoprecipitated using anti‑Strep-beads and the indicated co-immunoprecipitated proteins were analyzed by Western blotting. β-Actin served as a loading control."
NCBI gene IDs:
"RIPK3: 11035"
Figure legend: "E USP22 KO HT-29 cells and USP22 KO cells re-expressing PAM-mutated 3xFLAG-HA-USP22 were pre-treated with 20 µM zVAD.fmk, 5 µM BV6 for 1 h. After pre-treatment, 10 ng/ml TNFα was added for 2 h, as indicated. 3xFLAG-HA-USP22 was immunoprecipitated using anti-HA-beads and the indicated co-immunoprecipitated proteins were analyzed by Western blotting. β-Actin served as a loading control."
NCBI gene IDs:
"FLAG: 
HA: 
USP22: 23326"
Figure legend: "A HT-29 control, USP22 KO and USP22 KO cells re-expressing PAM-mutated 3xFLAG-HA-USP22 WT or C185S were stimulated with 20 µM zVAD.fmk, 0.5 µM BV6 and 1 ng/ml TNFα for 4 h. Poly-ubiquitinated proteins were enriched by GST-TUBE pull-down, followed by incubation with the catalytic domain of USP2, as indicated. RIPK3 and USP22 expression and levels of ubiquitinated RIPK3 were monitored using Western blotting with the indicated antibodies. β-Actin served as loading control. Ponceau-staining was used to confirm equal loading of GST-TUBE."
NCBI gene IDs:
"FLAG: 
HA: 
USP2: 9099
USP22: 23326"
Figure legend: "B HT-29 control and USP22 KO cells were transfected with His-ubiquitin for 24 h, as indicated. Cells were pre‑stimulated with 20 µM zVAD.fmk, 0.5 µM BV6 for 1 h. Following pre-treatment, 1 ng/ml TNFα was added for 4 h. His-ubiquitin was immunoprecipitated using Ni-NTA beads and detection of indicated proteins was performed by Western blotting. β-Actin served as loading control for the input, whereas His-ubiquitin levels served as loading control for immunoprecipitated ubiquitin."
NCBI gene IDs:
"USP22: 23326"
Figure legend: "C HeLa TRex RIPK3 control and USP22 KO cells were incubated with 1 µg/ml Dox and transfected with HA-ubiquitin for 24 h, as indicated. Cells were pre-stimulated with 20 µM zVAD.fmk, 5 µM BV6 for 1 h. Following pre-treatment, 10 ng/ml TNFα were added for 3 h. HA-ubiquitin was immunoprecipitated using anti-HA-beads and detection of indicated proteins was performed by Western blotting. β-Actin served as loading control for the input, whereas HA-levels served as loading control for immunoprecipitated ubiquitin."
NCBI gene IDs:
"USP22: 23326"
Figure legend: "B Bar graphs demonstrating the number of up‐, non‐, and downregulated ubiquitination sites in USP22 KO HT-29 cells versus control under TBZ-treatment."
NCBI gene IDs:
"USP22: 23326"