EMBO/soda-nel huggingface dataset:
Activation of autophagy by inflammatory signals limits IL-1β production by targeting ubiquitinated inflammasomes for destruction (2012)
figf8

(a) Immunoblot analysis of lysates of LPS (20 ng/ml)–primed mouse BMDMs exposed to LPS (500 ng/ml) and ATP (3 μM), poly(dA:dT) (1.5 μg/ml) or uric acid crystals (50 μg/ml), detecting GTP-bound RalB (collected by RaBP1-agarose affinity purification) and RalB in total lysates. (b) Immunoblot analysis of lysates and supernatants of mouse BMDMs transfected with control or RalB-specific siRNA on 2 sequential days, then primed with LPS and exposed for 6 h to poly(dA:dT) (1.5 μg/ml), probed for RalB, LC3 and IL-1β; supernatants were collected after overnight culture in serum-free medium (numbers below lanes as in Fig. 1d). Nonsp, nonspecific band. (c) Immunoblot analysis of lysates of wild-type and Aim2−/− mouse BMDMs transfected with control or AIM2-specific siRNA on 2 sequential days, primed for 2 h with LPS and then exposed for 1 h to poly(dA:dT) (numbers below lanes as in Fig. 1d). (d) Immunoblot analysis of ASC immunoprecipitates from LPS-primed mouse BMDMs left untreated or transfected for 1 h with poly(dA:dT) and treated for 30 min with LPS and ATP. Mock, immunoprecipitation with control antibody. (e) Confocal microscopy of LPS-primed mouse BMDMs left untransfected or transfected for 30 min with poly(dA:dT), immunostained for RalB and AIM2 (left three columns); RalB and ASC (middle columns); and beclin-1 and AIM2 (right two columns). Bottom row, 3× electronic enlargement of areas outlined above. Scale bars, 10 μm. Data are representative of at least two experiments.
KRAB zinc finger proteins interactome reveals the evolution of their functional diversification (2019)
Figure 1

Figure 1: KZFPs IFs and interactome

A. IF by confocal microscopy performed on KRAB-HA overexpressing cells. Staining was performed with anti-HA (Alexa-488, green), anti-HSPA5 (Alexa-647, red) and DNA was stained with Dapi (blue). B. Force-directed network representing detected interactions. Each bait KZFP is represented by a blue circle ("node") and linked to green nodes that represent its preys. Specific preys, such as KAP1 is represented in red and its associated proteins SIRT1, SMARCAD1 and HP1α, HP1γ in orange. All interactions represented are below the false discovery rate of 1%. The topology of the network is established by a force-directed process that follows certain rules: all nodes repel each other and are attracted to the center by artificial "gravity" and nodes with links attract each other. Weighted links between any nodes are based on the average fold change over controls. C. Zoom-in on KAP1 core of the interactome. D&E. Zoom-in on KZFPs enriched with unique interactors. F. Zoom-in on the subset of KZFPs not connected to the main interactome.
KRAB zinc finger proteins interactome reveals the evolution of their functional diversification (2019)
Figure 2

Figure 2: SCAN and DUF3669 domains are involved in oligomerization

A. Force-directed network representing the SCAN interactome displaying SCAN-containing baits and their SCAN-containing preys. B. HA immunoprecipitation of stably expressed ZNF282-HA in cells previously transfected with ΔDUF3669-ZNF398-GFP and WT-ZNF398-GFP followed by detection of ZNF398 constructs in the IPs through Western-Blot using an anti-GFP antibody. Bottom: Western-Blot using an anti-HA antibody on the IPs. Input = Cellular lysate, IP = Immunoprecipitate C. Left: DUF3669-only and KRAB domain constructs used. Right: HA immunoprecipitation in cells previously co-transfected with ZNF398-DUF3669-HA and either ZNF282-KRAB-Flag or ZNF282-DUF3669-Flag followed by detection of either of these protein constructs in the IPs through Western-Blot using an anti-Flag antibody. Bottom: Western-Blot using an anti-HA antibody on the IPs.
KRAB zinc finger proteins interactome reveals the evolution of their functional diversification (2019)
Figure 6

Figure 6: KZFPs present in the last common ancestor of coelacanth and tetrapods were KAP1 binders

A. Consensus sequences for KRAB A-Boxes from top to bottom: coelacanth KRAB domains, coelacanth PRDM9 KRAB domain, human PRDM9 KRAB domain and human KRAB domains. Residues conserved between the first three sequences and not present in human KRAB consensus were highlighted in red. In the human KRAB consensus, residues crucial for KAP1 recruitment were highlighted in blue. B: Immunoprecipitations of HA-tagged KRAB domains in order to check  interaction with cKAP1. Co-transfection of Flag-tagged cKAP1 (upper panel) or Flag-tagged hKAP1 (lower panel) and HA-tagged ZNF398 DUF3669 domain negative control, H2ZYM4 and M3XKG1 cKRAB domains and ZNF93 hKRAB domain in KAP1 KO HAP1 cells followed by HA-immunoprecipitation. cKAP1 and hKAP1 presence was revealed by Western-Blot using an anti-Flag antibody. Input = Cellular lysate, IP = Immunoprecipitate. Western-Blot using an HA antibody on the IPs at the bottom. C. Evolutionary KRAB model: On top, a simple phylogenetic tree represents the links between echinoderms, coelacanth and human. At the bottom different KRAB domain versions ranging from the ancestral PRDM9-related KRAB domain to the classical KAP1 binding human KRAB are represented. Their position on this model depends i) under which species or clades they were detected, hence reflecting the last common ancestor in which they putatively appeared and ii) whether they bind KAP1, in the blue rectangle, or not, in the pink rectangle. Briefly, the echinoderm-conserved PRDM9 related KRAB domain is the putative ancestor of KAP1-binding KRAB domain that emerged in the coelacanth. The KRAB domain and KAP1 co-evolved, maintaining their association in tetrapods. Meanwhile, certain KZFPs have lost KAP1 binding to become vKZFPs.
Open conformation of tetraspanins shapes interaction partner networks on cell membranes (2020)
Figure 3

Figure 3. CD53-CD2 and CD81-CD19 partner interactions identified by HDX-MS and modeled onto open and closed conformations.

A The partner-binding regions of CD53 and CD81. Their EC2 sequences are aligned. Identical residues are shaded in dark grey, similar residues in light grey, and partner-binding regions identified by HDX-MS in orange. Secondary structures are indicated above.

B Representative HDX-MS curves showing the protected regions in CD53 and CD2. Other curves at these regions are shown in Figure EV4A and B, and complete HDX-MS results in Fig 3 Source Data. The HDX experiments were performed (manually or with a LEAP automation system) in triplicate. Error bars show the standard deviation.

C Left, CD53-CD2 docking model. The HDX-MS identified regions are shown as a red rod in CD53 and as a blue arrow in CD2. The black box indicates the CD2 transmembrane region and the dashed line indicates the linker to Ig domains. Right, CD53-CD2 docking model with CD53 modeled in the closed conformation. The modeling was based on the CD81 structure, in which EC2 reorients to interact with the TM domain. Docking of CD19 Ig to CD53 EC2 is in the same manner as Left.

D Representative HDX-MS curves showing the protected regions in CD81 and CD19. Other curves at these regions are shown in Fig EV4C and D, and complete HDX-MS results in Fig 3 Source Data. The HDX experiments were manually performed in triplicate. Error bars show the standard deviation.

E Left, CD81-CD19 docking model in the closed conformation. The HDX-MS identified binding regions are colored as in C. Right, Modeled open conformation of CD81 and docking with CD19 (in the same manner as Left). MD simulation (1 µs) of this open conformation is shown in Fig 4 and Movie EV1.
Open conformation of tetraspanins shapes interaction partner networks on cell membranes (2020)
Figure 5

Figure 5. Accurate glycosylation of CD19 requires the EC1-supported conformation of CD81.

A CD81 knockout of 293T cells. The knockout cell line was generated by CRISPR-Cas9. The immunoblots using CD81 antibody and actin antibody were conducted on the same film.

B CD81 facilitates CD19 maturation. CD81 and CD19 (300 ng DNA) were transfected into the CD81-knockout 293T cells. Anti-FLAG immunoblot was performed for CD19 with a C-FLAG tag. The deglycosylation was performed by digesting the cell lysate with PNGase F or Endo H, following the manufacturers' instructions. The deglycosylation pattern of CD19 species (p: precursor, m and m': two mature forms) differs because the glycosylation content of these species is different (see text for explanation).

C Trypsin digestion of CD19. The digestion was performed at 37 °C with intact cells. The mature CD19 forms (m and m') can be digested because they are surface expressed, whereas the intracellular precursor form (p) is protected from the digestion.

D EC1-EC2 interaction in the CD81 open conformation (modeled as in Fig 3E, right). The EC2 hydrophobic groove is shown in surface representation, and the interacting EC1 residues are indicated.

E EC1 mutants of CD81 interfere with the accurate glycosylation of CD19. Chol: mutations at the cholesterol-binding site of CD81. CD81 constructs and CD19 (300 ng DNA) were co-transfected into the CD81-knockout 293T cells. Top, Anti-FLAG immunoblot was performed for CD19 with a C-FLAG tag. In presence of wildtype CD81 (wt), most CD19 is in the properly glycosylated mature form (m), and the overglycosylated m' form is not observed. With the EC1 mutants (A4 in particular), however, m' emerges and m shifts to high MW, indicating an alteration in the CD19 glycosylation. The level of intracellular p form is low with the wildtype CD81 or with the mutants. Bottom, Control experiment showing similar expression level of wild-type CD81 and CD81 mutants. Anti-V5 immunoblotting was conducted for the CD81 constructs with a C-V5 tag, and anti-actin immunoblotting was conducted on the same film.
Open conformation of tetraspanins shapes interaction partner networks on cell membranes (2020)
Figure 6

Figure 6. CD53 promotes the migration of pre-B cells and disruption of the CD53 open conformation impairs the migration.

A Migration of naïve Cd53-/- cultured primary pre-B cells to 100 ng/mL CXCL12 compared to WT littermate controls (n = 5 mice per group) over three independent experiments. Bars and error bars represent mean ± standard error of the mean. **p < 0.01 by two-way ANOVA.

B Migration of Cd53-/- pre-B cells transduced with empty, control, or mutant constructs in response to CXCL12 gradient, 3 days after transduction (n = 2-5 mice per group) over two independent experiments. Bars and error bars represent mean ± standard error of the mean. **p < 0.01 and ***p < 0.001 by two-way ANOVA.

C Regulation of tetraspanin-partner interaction by open and closed conformational changes. The cartoon shows the mushroom fold of EC2 (stalk and head) and supporting interaction of EC1 (green). The spiral roots represent TMs. Left, Partner interaction at the open conformation requires EC2 in an exposed orientation and its C-D variable region at an above-membrane height (double arrow) matching that of the partner Ig domains. Right, Without the EC1 support (dimmed), changes in the EC2 orientation and C-D height prevent partner interactions in the closed conformation.

Expanded View Figure legends
Pseudomonas syringae Senses Polyphenols via Phosphorelay Crosstalk to Inhibit Virulence (2021)
Figure 2

Figure 2. Polyphenols suppress bacterial virulence by abolishing RhpS

(A, B and C) Polyphenols inhibits the autophosphorylation of RhpS-FL protein. Inverted membrane vesicles containing RhpS-FL protein were phosphorylated with 10-μCi [γ-32P]-ATP. Polyphenols TA (A), PGG (B), or EGCG (C) were added into the mixture for 20 min at different concentrations before stopping the reaction. The phosphorylated proteins were separated by 12% SDS-PAGE. After electrophoresis, the SDS-PAGE gels were dried with gel-dryer and then exposed to a medical X-ray film overnight. This experiment was repeated twice, yielding similar results.

(D, E and F) Polyphenols inhibit the phosphatase activity of RhpS. The GST-RhpR was phosphorylated by adding 5-μM PSPPH_3550c protein as well as 10-μCi [γ-32P]-ATP for 10 minutes. The RhpS-FL protein was treated in the presence of different concentrations of TA (D), PGG (E), or EGCG (F) for 10 min. Then, the full-length RhpS protein was added into the mixture phosphorylated GST-RhpR for 1 hour before stopping the reaction. The phosphorylated proteins were separated by 12% SDS-PAGE. After electrophoresis, the SDS-PAGE gels were dried with gel-dryer and then exposed to a medical X-ray film overnight. This experiment was repeated twice, yielding similar results.

(G) Quantification of the band intensities of (D, E and F). The intensities of the phosphorylation bands were calculated using Image J software. The individual data points were shown (n=2 biological replicates).

(H) Venn diagram of TA/PGG/EGCG-inhibited genes and RhpS-activated genes at 4-fold cut-off.

(I, J and K) Polyphenols inhibits the virulence levels and in planta growth of Psph in bean plants. Bacterial virulence levels were estimated by inoculating Psph strains with or without polyphenols onto host bean plants. Disease symptoms were recorded 5 days after inoculation. Leaf disks (1 cm2) were removed 5 days after inoculation and diluted in sterile water to count bacterial CFUs. The experiment was repeated three times.

(L) Ion leakage assay of Pto DC3000 infection of bean plants with or without indicating polyphenols. The error bars represent standard deviations (n=3 biological replicates).

Data information: In (A to F), the reaction was stopped by adding 5 × loading buffer before SDS-PAGE. Abbreviation is as follows: CBB, coomassie bright blue stain; CFU, colony-forming unit. In (I, J, K and L), * indicate significant differences between mock group and strains under the different test conditions as indicated. (Student's t test. *, P ≤ 0.05, **, P ≤ 0.01, and ***, P ≤ 0.001). Error bars represent standard deviations (n=3 biological replicates).
Pseudomonas syringae Senses Polyphenols via Phosphorelay Crosstalk to Inhibit Virulence (2021)
Figure 3

Figure 3. The Pro40 residue of RhpS is required for polyphenols-RhpS interaction

(A) Schematic view of protein structure of RhpS.

(B, C and D) Polyphenols bind to the RhpS-FL protein. The concentration of RhpS-FL protein was consistently measured as 0.6-μM. The binding curves of TA (B), PGG (C) and EGCG (D) are shown. The individual data points were shown (n=2 biological replicates).

(E) Alanine-scanning mutagenesis of the RhpS sensor. RhpS containing a corresponding mutation was provided in the rhpS deletion strain on a pHM1 vector. LUX activity was estimated in the absence (black frame) and presence TA (50-μM), PGG (100-μM) or EGCG (2-mM). Error bars represent standard deviations (n=3 biological replicates).

Data information: In (E), * indicate significant differences between mock group and strains under the different test conditions as indicated. (Student's t test. *, P ≤ 0.05, **, P ≤ 0.01, and ***, P ≤ 0.001).
Pseudomonas syringae Senses Polyphenols via Phosphorelay Crosstalk to Inhibit Virulence (2021)
Figure 4

Figure 4. PSPPH_5115, PSPPH_3736 and PSPPH_3550 cooperatively act as kinases on RhpR in vitro

(A) GST-RhpR or His6-RhpR was phosphorylated by RhpSc, PSPPH_3550c, PSPPH_3736c or PSPPH_5115c proteins. 10-μCi [γ-32P]-ATP was mixed with or without GST/His6-RhpR and incubated with reaction components for 20 minutes. The reaction was stopped by adding 5 × loading buffer before SDS-PAGE. The phosphorylated proteins were separated by 12% SDS-PAGE. After electrophoresis, the SDS-PAGE gels were dried with gel-dryer and then exposed to a medical X-ray film overnight. This experiment was repeated twice, yielding similar results.

(B) The results from (A) were quantified by Image J. The autokinase and kinase activities of HKs were compared with that of RhpS. The individual data points were shown (n=2 biological replicates).

(C and D) The rhpS/PSPPH_3550/PSPPH_3736/PSPPH_5115 quadruple deletion strain restored the T3SS induction of rhpS deletion strain. The wild type, rhpS deletion and quadruple deletion strains were cultured in MM (C) for 6 hours or KB (D) to OD600=0.6. Transcripts of the indicated genes were determined by RT-qPCR.

(E) The activity of rhpR-lux reporter in the wild-type, rhpS deletion and quadruple deletion strains.

(F) The quadruple mutant strain restored the pathogenicity of rhpS deletion strain. Bacterial virulence levels were estimated by inoculating wild type, rhpS deletion and quadruple deletion strains onto host bean plants. Disease symptoms were recorded 5 days after inoculation. The experiment was repeated three times.

(G) Leaf disks (1 cm2) from (G) were removed 5 days after inoculation and diluted in sterile water to count bacterial CFUs. The experiment was repeated three times.

Data information: In (C, D, E and G), * indicate significant differences between rhpS mutant and quadruple mutant. Error bars represent standard deviations (n=3 biological replicates). Student's t test. *, P ≤ 0.05, **, P ≤ 0.01, and ***, P ≤ 0.001.
The nucleoporin Nup50 activates the Ran guanyl-nucleotide exchange factor RCC1 to promote NPC assembly at the end of mitosis (2021)
Figure 1

Figure 1: Nup50 is crucial for NPC assembly

(A) Western blot analysis of mock and Nup50 depleted Xenopus egg extracts, with or without addition of recombinant Xenopus Nup50. 1, 2, and 4 µl of extracts were analyzed with indicated antibodies.

(B) Confocal microscopy images of fixed nuclei assembled for 120 min in mock depleted (mock) and Nup50 depleted (∆Nup50) Xenopus egg extracts supplemented with either buffer or recombinant Nup50. In the left column, membranes were pre-labelled with DiIC18 (1,1'-Dioctadecyl-3,3,3',3'-Tetramethylindocarbocyanine Perchlorate, red) and chromatin was stained with DAPI (4′,6-Diamidin-2-phenylindol, blue). Three right columns show the immunofluorescence staining for Nup50 (green) and, NPCs (mAB414, red) on the chromatin (DAPI). Scale bars: 10 µm.

(C) The average percentage of closed nuclear envelopes (upper panel) as well as mAB414 positive nuclei (lower panel) for 100 randomly chosen chromatin substrates in each of 3 independent experiments is shown. Data points from individual experiments are indicated.

(D) HeLa cells were transfected with 10 nM control (n=84) or Nup50 siRNA (n=171), 20 nM control (n=101) or Nup50 siRNA (n=157) and 40 nM control (n=86) or Nup50 siRNA (n=204). 72 h post transfection, cells were fixed with 4% PFA and stained with antibodies against Nup50 and mAB414, chromatin was labeled with DAPI. Scale bars: 50 µm. Quantitation of the mAB414 rim intensity at three different Nup50 RNAi and control oligo concentrations. The means are indicated as diamonds, error bars show the standard deviations. P-values have been calculated from a student t-test comparing the mean between the experimental conditions.

(E) Western blot analysis of HeLa cells treated as in (D) 72 h post transfection.
The nucleoporin Nup50 activates the Ran guanyl-nucleotide exchange factor RCC1 to promote NPC assembly at the end of mitosis (2021)
Figure 2

Figure 2: Two Nup50 paralogues in mouse cells

(A) Mouse 3T3-NIH cells were transfected with 20 nM control, Nup50A, Nup50B or a combination of Nup50A and Nup50B siRNA. After 72h cells were fixed and stained with mAB414 (green) and antibodies against mouse Nup50 (red). Chromatin is stained with DAPI (blue). The merge of the three channels is shown on the left column. Scale bar: 50 µm. An insert shows a zoom on a representative nucleus for each picture. Scale bar: 10 µm.

(B) Quantitation of the mAB414 rim intensity from n=4 independent experiments done as in (A), each represented by a different color. The mean of each individual experiment and condition is represented by a diamond, the mean of all four experiments by a horizontal black line. P-values have been calculated from a paired ratio t-test comparing the mean between the experimental conditions. Error bars show the standard deviations of the means.

(C) Western blot showing the amount of Nup50A (top row) for each of the experimental condition. The actin (lower row) is shown as a loading control.
The nucleoporin Nup50 activates the Ran guanyl-nucleotide exchange factor RCC1 to promote NPC assembly at the end of mitosis (2021)
Figure 3

Figure 3: Nup50 and MEL28 do not depend on each other for chromatin binding

(A, B) Demembranated sperm chromatin was preincubated in Xenopus egg extract, depleted for MEL28/ELYS or Nup50 (B) or control treated (Mock, A). After 10 min, membranes were added to the reaction. Reactions were stopped at the indicated time points by fixation and analyzed by confocal microscopy after immunostaining with α-MEL28/ELYS (A), α-Nup50 (B), and mAb414 (A, B). Chromatin was stained with DAPI (blue in the overlay). Scale bar: 10 μm.
The nucleoporin Nup50 activates the Ran guanyl-nucleotide exchange factor RCC1 to promote NPC assembly at the end of mitosis (2021)
Figure 4

Figure 4: Nup50 is a chromatin binding protein

3 µM recombinant EGFP, EGFP-tagged MEL28/ELYS (aa 2290-2408) or EGFP-tagged Nup50 were incubated with empty or DNA-coated magnetic beads, which were chromatinized with Xenopus egg extracts (right panel) or unchromatinized DNA-beads (left panel). After 3h the beads were re-isolated, washed, co-stained with DAPI and analyzed by confocal microscopy. Scale bar: 10 µm.
The nucleoporin Nup50 activates the Ran guanyl-nucleotide exchange factor RCC1 to promote NPC assembly at the end of mitosis (2021)
Figure 5

Figure 5: A minimal 46 aa region is required for Nup50 NPC localization

(A) HeLa cells were transfected with EGFP-Nup50 and different truncations. After 24h cells were shortly pre-treated with 0.1% Triton X-100 in PBS, fixed with 4% PFA and analyzed by confocal microscopy (left panel). Scale Bar: 10 µm. The graph (right panel) shows the Nup50 domains (blue: Importin α interaction domain, green: NPC targeting domain and pink: Ran binding domain) and the truncation tested. Ability to bind the nuclear rim is indicated on the right.

(B) Sequence alignment of Nup50 144-189, species are indicated on the left, the black boxes highlight residues tested by single point mutation in (C) and (D), and the residues number are shown above or below the alignment. The color scheme indicates the type of amino acids according to the alignment software default setting.

(C) HeLa cells were transfected with EGFP-Nup50 comprising different point mutations in the minimal NPC binding region and analyzed as in (A). Scale Bar: 10 µm.

(D) GST-fusion constructs of the Xenopus Nup50 minimal NPC binding fragment (aa 144-189) comprising no or single point mutations were incubated with Xenopus egg extracts. Starting material as well as bound proteins were analyzed by Western blotting with indicated antibodies. The quantitation shows the average MEL28/ELYS and Nup153 bead bound signal from three independent experiments, normalized to the signal of their respective wild-type GST fusion (see Appendix Figure S1D for a gel showing the GST-baits). Data points from individual experiments are indicated.
The nucleoporin Nup50 activates the Ran guanyl-nucleotide exchange factor RCC1 to promote NPC assembly at the end of mitosis (2021)
Figure 6

Figure 6: A N-terminal Nup50 fragment is required for NPC assembly

(A) Confocal microscopy images of nuclei assembled for 120 min in mock depleted, Nup50 depleted (∆Nup50), and Nup50 depleted Xenopus egg extracts supplemented with recombinant wild-type Nup50 or different mutants. Nuclei were fixed in 4% PFA and 0.5 % glutaraldehyde, stained for NPCs (mAB414) and the chromatin (DAPI). Scale bar: 10 µm.

(B) Average percentage of mAB414 positive nuclei for 100 randomly chosen chromatin substrates in each of at least three independent experiments shown in (A). Data points from the individual experiments are indicated.

(C) Confocal microscopy images of nuclei assembled with N-terminal Nup50 truncations and an minimal N-terminal fragment. Samples were analyzed as in (A).

(D) Average percentage of mAB414 positive nuclei for 100 randomly chosen chromatin substrates in each of at least three independent experiments shown in (C). Data points from the individual experiments are indicated.