EMBO/soda-nel huggingface dataset:
Mpl Traffics to the Cell Surface Through Conventional and Unconventional Routes (2014)
Figure 7

Surface Mpl is rapidly internalized after Tpo addition, followed by recovery from recycling and autolysosome compartments.

A) Western blot analysis and quantification of streptavidin-immunoprecipitated surface-biotinylated proteins, probed with an anti-Mpl antibody. B and C) Confocal images of K562 cells transiently expressing MplmOrange2. Cells in (B) are untreated. Cells in (C) were stimulated 60' with Tpo, washed and chased for 15?min before imaging. D) Quantification of plasma membrane fluorescence in resting and Tpo-stimulated cells. Mean fluorescence at the membrane was estimated by averaging the total fluorescence after values equal to or below background. A two-tailed Student t-test shows a significant difference in the membrane fluorescence after stimulation with Tpo followed by Tpo removal. Scale bars?=?5?µm. E) Western blot analysis of protein phosphorylation in HEL, K562Mpl-mKO2 and K562Mpl-mOrange2 cells after Tpo stimulation (50?ng/mL Tpo, 10?min).
Mpl Traffics to the Cell Surface Through Conventional and Unconventional Routes (2014)
Figure 8

Complex Mpl trafficking includes conventional and autophagic secretory pathways, as well as multiple recycling pools.

A and B) Surface biotinylation and total lysates western blot analysis of HEL or K562Mpl-mKO2 cells after treatments for 60?min with Tpo, ionomycin (I), PMA, Mon or A23187. C) Total lysate, surface biotinylation and exosomal fraction western blot analysis of HEL and K562Mpl-mKO2 cells treated with Mon or A23187 for 15?h. D) Western blot analysis of HEL cells treated, or not, with Bafilomycin A1 at 100?nm for 3?h and then stimulated or not with Tpo at 25?ng/mL for 30?min. The quantification of the blot is shown. E) Surface biotinylation and total lysates western blot analysis of HEL cells after treatments for 30?min with Tpo or for 3?h with 3-MA (3-Methyladenine) or Rp. F) Total lysate and surface biotinylation western blot analysis of HEL cells overexpressing ATG5 K130R-V5 6?h post-transfection. G) Total lysate and surface biotinylation western blot analysis of HEL cells overexpressing either GRASP55-V5 or HA-STX5.
Membrane scission driven by the PROPPIN Atg18 (2017)
Figure 1

Figure 1. Redundancy of PROPPINs in vacuole fission.

a, Cells (wt, ∆atg18 or ∆atg18/∆atg21/∆hsv2 ()), with and without a plasmid expressing wt or a mutant form of Atg18) were grown in YPD, vacuoles were stained with FM4-64 and imaged by confocal microscopy. At least 10 confocal sections spaced at 300 nm were assembled into maximum projections. Images show cells before (0 min) and 15, 30 and 60 min after induction of vacuolar fragmentation by addition of 0.4 M NaCl. Red: FM4-64. Gray: DIC. Scale bar 5 µm. b, The number of vacuoles per cell was quantified for the experiment in a. 150 cells from three independent experiments were counted for each condition. Values represent the mean, error bars the SEM.
Membrane scission driven by the PROPPIN Atg18 (2017)
Figure 2

Figure 2. Localization of Atg18-GFP during salt-induced vacuole fission.

atg18 cells expressing plasmids carrying Atg18-GFP or Atg18FGGG-GFP, a mutant in the lipid binding sites, were stained with FM4-64. a, Confocal z-stacks were taken before and 2, 6 and 10 min after addition of 0.4 M NaCl. Stacks were processed into maximum projections using ImageJ. Scale bar: 5 µm. b, Same experiment as in a, but in a fab1 or vps34 background. c, The cells were categorized as indicated and the phenotypes of 150 cells were quantified. d, Localisation of Atg18-GFP 5 min after salt shock. Experiment as in a. Scale bar: 1 µm. e, Line scan analysis of fluorescence intensities in b. Red: FM4-64. Green: Atg18-GFP. Scale bar: 1 µm.
Membrane scission driven by the PROPPIN Atg18 (2017)
Figure 4

Figure 4: Lipid-triggered folding of the amphipathic helix of CD-loop peptides and its requirement for vacuole scission.

a, CD loop folding in hydrophobic buffer. Peptides were synthesized that correspond to the predicted amphipathic helix from S. cerevisiae Atg18 (TRLAREPYVDASRKTMGRMIRYSSQ) or to a sequence with reduced tendency to form amphipathic helices, generated by a swap of two amino acid pairs (indicated by arrows; SLoop). Helical wheel projections of the sequences are shown. b,c Their secondary structure was analyzed by circular dichroism spectroscopy in the presence of increasing concentrations of hexafluoro-2-propanol (HFIP), in the presence of 6 mM small unilamellar vesicles (SUVs, 70% egg PC and 30 % PS, total phospholipid concentration approx. 6 mM), or of or control buffer only (blue). Spectra are shown for b, the wildtype peptide, and c, the SLoop peptide. d, Vacuole morphology of cells expressing Atg18Sloop-GFP, imaged before and after osmotic shock as in Fig 1a. Scale bar: 5 µm. e, Number of vacuoles per cell in d, assayed as in Figure 1b. n=3. f, Vacuole morphology of cells expressing Atg18DLoop-GFP, imaged as in d. g, Number of vacuoles per cell in e, assayed as in Fig. 1b. n=3.
Membrane scission driven by the PROPPIN Atg18 (2017)
Figure 5

Figure 5: Binding of Atg18Sloop and Atg18Dloop to small unilamellar vesicles.

Binding of recombinant Atg18 variants to liposomes. SUVs were made of EPC with 5% of the indicated phosphoinositides, or of EPC and the indicated fraction of PS. Cholesterol had been added to 20 mol% of the total phospholipid amount. Liposomes were incubated with the purified proteins for 30 min at room temperature (25°C), centrifuged and the supernatants (S) and pellets (P) analyzed by SDS-PAGE and Coomassie staining. Binding is shown for a, Atg18wt; b, Atg18FGGG; c, Atg18DLoop; d, Atg18SLoop. e, The bands from a-d were quantified from 3 independent experiments. The mean with SEM is shown.
Membrane scission driven by the PROPPIN Atg18 (2017)
Figure 6

Figure 6. Different functional aspects of Atg18 displayed in vacuole fission and autophagy

a. Autophagy measured with the pho8∆60 assay in atg18 cells complemented with wt, FGGG, SLoop or DLoop alleles of ATG18. b. Same assay as in a, but in atg18 atg21 cells complemented with wt or SLoop versions of ATG18 and ATG21. Atg21SLoop contains K426F and F429K substitutions. Helical wheel projections for both Atg21 helices are given. The arrows indicate the magnitude of the hydrophobic moment. c. Vacuole fragmentation in atg2 cells, assayed 15 min after a moderate hypertonic shock as in Fig. 1. d. Number of vacuoles per cell in c, assayed as in Figure 1b. n=3.
Membrane scission driven by the PROPPIN Atg18 (2017)
Figure 7

Figure 7. Time lapse analysis of the effects of Atg18 on giant unilamellar vesicles.

a, Time lapse analysis of Atg18 added to liposomes. 10 µL of 10 µM purified recombinant Atg18wt was added to 100 µL buffer containing GUVs with 5% PI3P and 1% PI(3,5)P2. The sample was imaged on a spinning disc confocal microscope. Most of the tubules underwent scission into small vesicles (See movie EV6). Scale bar 5 µm. Areas in boxes 1 and 2 of c are shown at higher magnification in b and c. Scale bars: 2.5 µm. d, Quantification of fission activity. For each movie, the whole frame was divided into squares of 10 per 10 µm and the number of small vesicles per 100 µm2 counted. The mean of three independent experiments is shown with SEM.
Membrane scission driven by the PROPPIN Atg18 (2017)
Figure 8

Figure 8. Effect of Atg18 mutants on GUVs of different lipid composition

a, 1µM Atg18 was added to wells containing sedimented GUVs with 10% PI3P or PI(3,5)P2 while imaging a single plane by spinning disc microscopy. Membrane tension exerted by Atg18 binding suffices to induce liposome collapse, which allows to better judge the mobility of the tubular or clustered membranes that remain. Images are extracted from movies EV2 to EV5. Scale bar 5 µm. b, Atg18 was covalently labeled with the green fluorescent dye Alexa488 TFP (ThermoFisher) and used in experiments as in a, with rhodamine-phosphatidylethanolamine-labeled (Rh-PE) GUVs containing the indicated phosphoinositides. Scale bar 5 µm c, Effect of 1µM of purified recombinant Atg18 variants on GUVs containing 10mol% of the indicated phosphoinositides. Vesicles are shown after 1 to 3 min of incubation. d, Quantification of the experiments in c. The percentage of GUVs showing an enrichment of Atg18 flexible tabulated (arrowhead) or stiff aggregated structures (arrow) was determined. Shown are the mean and SEM from 3 independent experiments e, Quantification of the effect of different Atg18 version on the formation of tubules in c. Shown is the mean from 3 independent experiments with SD.
Membrane scission driven by the PROPPIN Atg18 (2017)
Figure 9

Figure 9. Oligomerization of Atg18

a, Cross-linking of Atg18. Purified recombinant Atg18wt or Atg18FGGG (1.5 µM) was incubated with liposomes containing 5% PI3P, PI(3,5)P2, PI(4,5)P2 or 15% PS as a control for negative charge. After addition of a cleavable cross-linker or buffer only, liposomes were pelleted, dissolved in sample buffer, incubated in the presence or absence of 100 mM dithiothreitol (DTT) for 5 min at room temperature and analysed by SDS-PAGE and Coomassie staining. Cross-link products migrate at the upper limit of the separating gel. b, Cross-linking experiment with recombinant Atg18wt as in a, performed in the presence of increasing concentrations of liposomes containing 5% PI(3,5)P2.
Glyoxalase 1 knock-down induces age-related β-cell dysfunction and
glucose intolerance in mice (2022)
Figure 4

Figure 4 - Immunohistochemistry evaluations and hormones measurement in pancreatic islets of 10-months old WT and Glo1KD mice.

A Pancreatic sections (20X magnification) from 10-months old WT and Glo1KD mice stained with hematoxylin and eosin (H&E), anti-insulin, anti-glucagon and anti-F4/80 antibodies, are representative of sections from 7 mice each group.

B, C Insular inflammatory infiltration indicated as incidence (% of infiltrated islets) (B) and severity score (C). Severity of infiltration has been graded as follow: absent (0), low (1), moderate (2) and high (3) (n=7 mice for each genotype).

D-G Morphometric analysis of islets indicated as the number of islets per sections (D) and the size of islets, calculated as the area (E), the major diameter (F) and the minor diameter (G) of the islets per mouse (n=7 mice for each genotype).

H, I and J Insulin and glucagon IS, calculated as the intensity score multiplied with percentage of positive cells (H, I) and the percentage of islets area stained with F4/80 (J) (n=7 mice for each genotype).

K, L Quantification of insulin (K) and glucagon (L) content in islet extracts by immunoenzymatic assay (n=7 WT mice vs n=10 Glo1KD mice).

M MGO content in pancreatic lysates by LC-MS/MS (n=6 mice for each genotype).

Data information: In (B-M), box plots show the minimum and maximum values (ends of the whiskers), interquartile range (length of the box), median (line through the box) and mean (+) values. *p≤0.05, **p≤0.01 (Mann-Whitney test).
Glyoxalase 1 knock-down induces age-related β-cell dysfunction and
glucose intolerance in mice (2022)
Figure 7

Figure 7 - Senolytics effect on MGO-treated INS-1 832/13 β-cell line.

A, B mRNA levels of Trp53 and Cdkn1a in INS-1 832/13 cells treated with MGO 0.05 mM for 48 hours or with MGO plus 24h of dasatinib 31 nM and quercetin 6 μM (D/Q) (n=6 biological replicates for each group). Gene expression is measured by qPCR as relative quantification to Cyclophilin A, used as reference gene.

C Whole cell protein lysates of INS-1 832/13 β-cells analysed by Western Blot with anti-p53 and anti-p21 antibodies; anti-vinculin antibody was used as loading control (n=9 biological replicates for each treatment group).

D Nuclear protein lysates of INS-1 832/13 β-cells analysed by Western Blot with anti-HMGB1 antibody; anti-histone H3 antibody was used as loading control (n=5 biological replicates for each treatment group).

E Proportion of β-Gal+ β-cells analysed by a fluorescence-based assay using flow cytometry (n=4 biological replicates for each treatment group).

F Cytokine levels measured in the β-cell culture medium by a multiplex immunoassay, are shown as fold increase over not treated control cells (n=6 biological replicates for each treatment group).

G GSIS measured in the β-cell culture medium by an ELISA assay. Insulin concentration is reported at basal condition (2.5 mM glucose) and in response to glucose stimulation (16.7 mM) (n=4 biological replicates for each treatment group).

Data information: Data in bar graphs are presented as mean ± SEM. In C and D, blots show representative image of replicate experiments; protein levels were quantified by the densitometric analysis of independent experiments and showed in the bar graphs as the percentage over not treated control cells. *p≤0.05, **p≤0.01, ***p≤0.001: MGO vs CTR; #p≤0.05, ##p≤0.01, ###p≤0.001: D/Q vs MGO (Mann-Whitney test in A-D and F, Student's t-test in E and G).

Expanded view figure legends
EGFR is required for FOS-dependent bone tumor development via RSK2/CREB signaling (2018)
Figure 1

Figure 1 - EGFR signaling is essential for c-Fos-dependent bone tumor formation.

A X-ray analysis of 6-months-old H2-c-fosLTR/Egfrwa/+ and H2-c-fosLTR/Egfrwa/wa littermates. Scale bars: 1cm.

B Bone tumor number per mouse at 5-6 months of age (n=23 wa/+, 15 wa/wa).

C Quantification of tumor size in tibiae at 5-6 months of age (n=23 wa/+, 15 wa/wa).

D Alkaline Phosphatase (ALP) levels in the serum at endpoint (age = 5-6 months; n=16 wa/+, 11 wa/wa).

E X-ray analysis before (age = 2 months) and after (age = 7 months) vehicle or Erlotinib treatment.

F Quantification of tumor number during treatment (n=6 Vehicle, 5 Erlotinib).

G Tumor size during treatment (n=6 Vehicle, 5 Erlotinib).

H Analysis of serum ALP levels during treatment (n=5).

I PET summation images (0-90 min) in horizontal (upper panel) and coronal view (lower panel) depicting [11C]Erlotinib distribution in one H2-c-fosLTR/Egfrwt mouse (M125). Anatomical structures are labelled with arrows (T, tumor; L, liver; H, heart; B, brain). Scale bars: 1cm.

J Concentration-time curves of [11C]Erlotinib in bone tumors in right scapula of three H2-c-fosLTR/Egfrwt mice measured with PET. Broken line indicates threshold for in vitro effect of Erlotinib (1 µM).

Data information: Data are shown as mean ± SEM. p-values were calculated by unpaired, two tailed t-test (B, C and D) or by two-way ANOVA followed by Bonferroni multiple comparisons test (F, G and H).
EGFR is required for FOS-dependent bone tumor development via RSK2/CREB signaling (2018)
Figure 2

Figure 2 - Osteoblast-specific EGFR deletion reduces c-Fos driven osteosarcoma formation.

A X-ray analysis of 7-months-old H2-c-fosLTR/Egfrwt and H2-c-fosLTR/Egfr∆Ob littermates. Scale bars: 1cm.

B Bone tumor number per mouse at 6-7 months of age (n=22 wt, 11 ∆Ob).

C Quantification of tumor size in tibiae (n=22 wt, 11 ∆Ob).

D ALP levels in the serum at 7-month endpoint (n=17 wt, 8 ∆Ob).

E µPET/CT analysis of 7-months-old H2-c-fosLTR/Egfrwt and H2-c-fosLTR/Egfr∆Ob littermates.

F Standardized uptake values (SUV) of the µPET-tracer Na[18F]F in the pelvic osteosarcoma of 4- and 7-months-old H2-c-fosLTR/Egfrwt and H2-c-fosLTR/Egfr∆Ob mice. n=6 wt, 4 ∆Ob for 4 month time-point, n=6 wt, 3 ∆Ob for 7 month time-point.

Data information: Data are shown as mean ± SEM. p-values were calculated by unpaired, two tailed t-test.
EGFR is required for FOS-dependent bone tumor development via RSK2/CREB signaling (2018)
Figure 3

Figure 3 - EGFR is essential for proliferation, survival and c-Fos protein and mRNA expression via RSK2/CREB phosphorylation.

A PCNA and cleaved Caspase 3 IHC staining and quantification shown as % positive cells (for PCNA) and as positive cells per mm² (for cleaved Caspase 3) in OS from H2-c-fosLTR/Egfrwt (n=6) and H2-c-fosLTR/Egfr∆Ob (n=5) mice. Scale bars: 100µm.

B Egfr, Ccnd1, c-fos and transgenic c-fos (c-fostg) mRNA expression levels in tumors of H2-c-fosLTR/Egfrwt and H2-c-fosLTR/Egfr∆Ob mice normalized to Tbp. n=17 wt, 14 ∆Ob (Egfr, Ccnd1), n=16 wt, 13 ∆Ob (c-fos), n=16 wt, 14 ∆Ob (c-fostg)

C IHC staining and quantification showing pRSK2 (n=4), pCREB (n=7 wt, 6∆Ob) and c-Fos (n=5) positive cells (%) in OS from H2-c-fosLTR/Egfrwt and H2-c-fosLTR/Egfr∆Ob littermates. Scale bars: 100µm.

Data information: Data are shown as mean ± SEM. p-values were calculated by unpaired, two tailed t-test.
EGFR is required for FOS-dependent bone tumor development via RSK2/CREB signaling (2018)
Figure 4

Figure 4 - EGFR controls c-Fos via MAPK-dependent CREB activation in primary OS cells.

A Western Blot analysis of primary OS cells isolated from H2‑c‑fosLTR/Egfrwt and H2-c-fosLTR/Egfr∆Ob mice.

B c-fos and transgenic c-fos (c fostg) mRNA expression levels in primary H2-c-fosLTR OS cells after 4x in vitro passages, cultured under standard conditions (n=3 independent cell lines).

C Western Blot analysis of H2-c-fosLTR/Egfrwt OS cells treated for 24h with Erlotinib.

D c-fos and c-fostg mRNA expression levels in H2-c-fosLTR/Egfrwt OS cells treated for 24h with Erlotinib (10µM) or DMSO as control (n=4 independent cell lines).

E, F Western Blot analysis of starved H2-c-fosLTR/Egfrwt OS cells, pre-treated with DMSO (1:1000), Afatinib (5µM), GSK2233470 (10µM), Rapamycin (10nM) or U0126 (10µM) for 30 minutes and stimulated with EGF (50ng/ml) as indicated.

G Western Blot analysis of primary OS cells isolated from a p53f/f Rb1f/f Osx-Cre mouse after 24h Erlotinib treatment.

H c-fos mRNA expression levels in p53f/f Rb1f/f Osx-Cre OS cells treated for 24h with Erlotinib (10µM) (n=3).

I Western Blot analysis of starved p53f/f Rb1f/f Osx-Cre OS cells, pre-treated with DMSO (1:1000) or Afatinib (5µM) for 30 minutes and stimulated with EGF (50ng/ml) as indicated.

Data information: Data are shown as mean ± SEM. p-values were calculated by unpaired, two-tailed t-test.
EGFR is required for FOS-dependent bone tumor development via RSK2/CREB signaling (2018)
Figure 5

Figure 5 - Osteoblast-specific overexpression of the EGFR ligand Amphiregulin accelerates tumor formation in H2-c-fosLTR mice.

A X-ray analysis of 6-months-old H2-c-fosLTR and H2-c-fosLTR/ColAREG littermates. Scale bars: 1cm.

B Bone tumor number per mouse at 5-6 months of age (n=22 wt, 13 ColAREG).

C Quantification of tumor size in tibiae at 5-6 months of age (n=22 wt, 13 ColAREG).

D Alkaline Phosphatase (ALP) levels in the serum at endpoint (age = 5-6 months; n=29 wt, 19 ColAREG).

E c-fos mRNA expression levels in osteosarcomas of H2-c-fosLTR (n=11) and H2-c-fosLTR/ColAREG mice (n=14).

F, G Western Blot analysis of bone tumor protein lysates from H2-c-fosLTR and H2-c-fosLTR/ColAREG mice.

Data information: Data are shown as mean ± SEM. p-values were calculated by unpaired, two-tailed t-test.
EGFR is required for FOS-dependent bone tumor development via RSK2/CREB signaling (2018)
Figure 6

Figure 6 - EGFR and c-Fos co-expression in human OS negatively correlates with patient survival.

A Representative images of OS biopsies stained with antibodies against EGFR and c‑Fos. Scale bars: 50µm.

B Kaplan-Meier survival curve comparing the survival of patients with EGFR and c-Fos double positive osteosarcomas against patients without co-expression of both proteins. (n=52).

C Gene expression correlation analysis of several cancer-associated RTKs with FOS in human OS (data analyzed from publicly available dataset E-GEOD-39058).

Data information: p-values were calculated by log-rank (Mantel-Cox) test comparing the two Kaplan-Meier curves (B) or by unpaired, two-tailed t-test (C).
EGFR is required for FOS-dependent bone tumor development via RSK2/CREB signaling (2018)
Figure 7

Figure 7 - EGFR inhibition decreases tumor progression of human OS cells 143b after orthotopic injection.

A Western Blot analysis showing EGFR and c-Fos protein levels in human OS cell lines cultured under standard conditions.

B Cell viability of 143b or LM7 cells cultured for 24h in medium (+10% FCS) with DMSO (1:2000) or Erlotinib (10µM) (n=3, representative result from 3 independent experiments, shown as fold change, normalized to DMSO).

C Cell viability of 143b or LM7 cells cultured for 24h in medium (+0.5% FCS) containing 1xPBS (contr., 1:200) or EGF (50ng/ml) (n=3, representative result from 3 independent experiments, shown as fold change, normalized to control).

D Treatment scheme: 143b OS cells (106 in 25µl PBS) were intratibially injected; mice were treated for 14 days with vehicle or Erlotinib (50mg/kg) 7 days after injection, when the tumors started to grow.

E 143b xenograft growth curve during therapeutic regime (n=8 vehicle, 6 Erlotinib; two independent experiments).

F 143b tumor weight at endpoint (n=8 Vehicle, 6 Erlotinib; two independent experiments).

G Treatment scheme: LM7 OS cells (106 in 25µl PBS) were intratibially injected; mice were treated for 21 days with vehicle or Erlotinib (50mg/kg) 42 days after injection, when the tumors started to grow.

H LM7 xenograft growth curve during therapeutic regime (n=7 Vehicle, 5 Erlotinib; two independent experiments).

I LM7 tumor weight at endpoint. n=7 vehicle, 5 Erlotinib, two independent experiments J IHC analysis of PCNA and cleaved Caspase 3 in 143b-derived tumors (n=4). Scale bars: 50µm.

K IHC analysis of pCREB and c-Fos in 143b-derived tumors. (n=5 for pCREB and n=4 for c-Fos analysis). Scale bars: 50µm.

L Western Blot analysis of pCREB/CREB and p-c-Fos/c-Fos protein expression in lysates directly isolated from 143b xenografts at endpoint, after 14 days of vehicle or Erlotinib treatment.

Data information: Data are shown as mean ± SEM. p-values were calculated by unpaired, two-tailed t-test (B, C, F, I, J and K) or by two-way ANOVA followed by Bonferroni multiple comparisons test (E and H).

EXPANDED VIEW FIGURE LEGENDS
DNA activates the Nse2/Mms21 SUMO E3 ligase in the Smc5/6 complex (2018)
Figure 2

Figure 2. Enhancement of the SUMO E3 ligase activity upon DNA binding by Smc5-Nse2 truncation complexes.

(A) Schematic representation of the domain composition of the heterodimeric full-length Smc5-Nse2 complex.

(B) Bar diagram representation of the relative SUMO conjugation activity of Nse2, full-length Smc5-Nse2, ΔHinge/Smc5-Nse2, ΔHead/Smc5-Nse2 and Arm/Smc5-Nse2 truncation constructs (schematic representation above). Orange bars indicate the presence of ssDNA (virion ϕx174) and red bars absence of ssDNA. Reaction rates were performed at least in three different independent experiments (see Fig EV2). Data values are mean ± s.e.m.; and n=3 technical replicates. Significance was measured by a two-tailed unpaired t-test relative to wild-type. **P<0.01.

(C) SYPRO-stained (left) and Western blot (right) time course SUMO conjugation reaction using and ΔHead/Smc5-Nse2 truncation construct in the presence of ssDNA. T7-tagged Smc5 and E1 were immunodetected by an anti-T7 antibody. Reactions were run at 30 °C and stopped at indicated times by adding SDS-loading buffer.

(D) SYPRO-stained (left) and Western blot (right) time course SUMO conjugation reaction using and ΔHinge/Smc5-Nse2 truncation construct in the presence of ssDNA. T7-tagged Smc5 and E1 were immunodetected by an anti-T7 antibody. Reactions were run at 30 °C and stopped at indicated times by adding SDS-loading buffer.

(E) Western blot of the time-course SUMO conjugation reaction in the presence of ssDNA (virion ϕx174) using either Nse2 (left) or Arm/Smc5-Nse2 complex (right). The reactions were run in the presence or absence of cNse4 external substrate. Reaction was run at 30°C and stopped at indicated minutes by adding SDS-loading buffer (N-S2, cNse4-SUMO2; N-2S2, cNse4-2SUMO2; N-3S2, cNse4-3SUMO2 and pS2, poly-SUMO2).