EMBO/soda-nel huggingface dataset:
A pH- and ionic strength-dependent conformational change in the neck region regulates DNGR-1 function in dendritic cells (2016)
Figure 6

Figure 6. Distinct parts of the neck region contribute to regulation of type-2 dimer formation

(A) Sequences of the neck region of human and long and short isoform of mouse DNGR-1 aligned in Clustal X software. Parts of the neck corresponding to the block deletions are depicted as green bars. (B) and (C) Supernatants containing the indicated ECD proteins (mouse, long isoform) were diluted 1:2 into the indicated buffers and analyzed by SDS-PAGE and Western blot under reducing or non reducing conditions. HRP-conjugated anti-FLAG antibody was used for detection of all proteins. Numbers on the side of blots indicate positions of molecular weight markers. Reduction-resistant dimers are indicated with an * and reduction-sensitive protein with a +. (D) Phoenix cells expressing the indicated proteins were lysed in 1% SDS in PBS or 10mM MES pH 6.1 buffers and the lysates were analyzed by SDS-PAGE and Western blot under reducing conditions. Anti-DNGR-1 antibody (clone 397) followed by HRP-conjugated secondary anti-rat antibody was used for detection. Numbers on the side of blots indicate positions of molecular weight markers. Reduction-resistant dimers are indicated with an * and reduction-sensitive protein with a +.
A pH- and ionic strength-dependent conformational change in the neck region regulates DNGR-1 function in dendritic cells (2016)
Figure 7

Figure 7. Type-2 dimer formation does not affect DNGR-1 ligand binding, coupling to Syk or receptor internalization

(A) Decreasing amounts of F-actin were spotted onto a nitrocellulose membrane and binding of indicated DNGR-1 ECD (mouse, long isoform) proteins was tested in PBS or MES pH 6.1 buffers. HRP-conjugated anti-FLAG antibody was used for detection. The signal was quantified by densitometry and quantitation of four independent experiments is shown. (B and C) B3Z-Syk reporter cells expressing indicated mutant or WT DNGR-1 receptors (mouse, long isoform) were incubated with UV-irradiated 293T cells (ratio dead:reporter cells is indicated), or with plate-bound anti-DNGR-1 antibody or medium alone at 37°C overnight. Activation of the NFAT reporter was measured at the end of the incubation period. Data are plotted as mean ± SD of experimental duplicates. One representative of three experiments is shown. (D) Phoenix cells expressing indicated mutant or WT DNGR-1 proteins (mouse, long isoform) were treated with F-actin, F-actin buffer, anti-DNGR-1 antibody or isotype-matched antibody of irrelevant specificity for 45 minutes, fixed in PFA, surface-stained for DNGR-1 and analyzed by flow cytometry. One representative of three experiments is shown.
A pH- and ionic strength-dependent conformational change in the neck region regulates DNGR-1 function in dendritic cells (2016)
Figure 8

Figure 8. The neck region of DNGR-1 controls its ability to promote cross-presentation of dead cell-associated antigens

(A) DNGR-1 KO MuTu DCs were transduced to express indicated DNGR-1 proteins, FACS-sorted and expanded. Surface (left panel) and total (right panel) DNGR-1 expression was determined by flow cytometry. (B and C) Ovalbumin-expressing mouse embryonic fibroblasts (bm1 T OVA MEFs) were UV-irradiated, left to undergo secondary necrosis and incubated overnight with MuTu cells transduced with indicated WT or mutant DNGR-1 proteins or an empty-vector control (ratio dead cells:MuTu cells is indicated) and OVA-specific pre-activated OT-I CD8+ T-lymphocytes. The amount of IFNγ accumulating in the culture medium was assessed using ELISA. One representative of two (B) or four (C) experiments is shown.
Lipase Maturation Factor 1 Affects  Redox Homeostasis in the Endoplasmic Reticulum (2018)
Figure 1

Figure 1: Identification of new LMF1-interacting partners.

A). Experimental schematic of DSP crosslinking experiment.

B). Schematic of pBPA crosslinking

C). Western blot of LMF1-partners after DSP crosslinking and affinity tag purification using LMF1's C-terminal His tag. Tetracycline induces LMF1 expression. Loading dye with 50mM DTT was used to break the disulfide bonds between LMF1 and its interacting partners. LMF1 complexes are labeled with asterisks and an arrow points to LMF1.

D). Western blot of LMF1-partners after pBPA photo-crosslinking and affinity tag purification. The DMSO panel does not include pBPA and serves as a negative control. Fractions were eluted with increasing concentrations of imidazole. An arrow points to LMF1.

E). SYPRO orange protein stain of DSP crosslinked samples. Samples without tetracycline but with DSP and DTT (negative control), and with tetracycline, DSP and DTT were sent for LC MS/MS. LMF1 complexes are labeled with asterisks and an arrow points to LMF1.

F). SYPRO orange stained gel of pBPA samples. An arrow points to LMF1.
Lipase Maturation Factor 1 Affects  Redox Homeostasis in the Endoplasmic Reticulum (2018)
Figure 2

Figure 2: siRNA knockdown of ERp44, ERp72, ERdj5, UGGT1, UGGT2 and TRX decreases LPL secretion.

A-B). HEK293 cells stably expressing LPL-V5 or PL-V5 were transfected with scrambled negative control siRNA (NC) or siRNA against LMF1-interacting partners. Media fractions were probed for the lipase V5 tags, and lysate fractions were probed for V5 and the LMF1 interacting-partners. GAPDH is a loading control.

C). siRNA knockdowns were performed in triplicate, and lipase secretion was quantified relative to NC siRNA. Error bars indicate the standard error.

D). Quantification of the percent of each protein remaining from three independent experiments. Error bars indicate the standard error.

E) HEK293 cells stably expressing LPL-V5 or PL-V5 were transfected with NC siRNA or siRNA against TRX. Lipase and TRX levels were tested as for Fig 1A.

F) Txnip overexpression in HEK293 cells stably expressing LPL-V5 or PL-V5 hinders LPL, but not PL, secretion.
Lipase Maturation Factor 1 Affects  Redox Homeostasis in the Endoplasmic Reticulum (2018)
Figure 3

Figure 3 LPL forms disulfide-bonded aggregates when LMF1 is missing.

A). A Western blot against the C-terminal V5 tags of both lipases shows that LPL, but not PL, forms intermolecular disulfide bonds in cells. Lipase monomers are marked with a single arrow, and aggregates are marked with double and triple arrows. The LPL aggregates are not present when samples are treated with DTT. There is no PL in the pellet fraction because PL does not aggregate.

B). A Western blot of a non-reducing gel of the lysate and pellet fraction of LPL grown in cells lacking LMF1 (HEK293∆LMF1), normal HEK293 cells or cells with additional LMF1 (+LMF1). LPL monomers and aggregates are marked as above.

C). Co-translational LPL folding was carried out in the presence of SP HEK293 or HEK293∆LMF1 cells. At the indicated time points, AMS was added to reactions to differentiate reduced (R) and oxidized (O) LPL. The position of the stacker layer is indicated.

D-F). The amount of reduced/total LPL intensity (D), the amount of oxidized/total LPL (E) and the intensity of the stacker/total LPL (F) was quantified. Each point is the average of three independent experiments, and error bars represent the standard error. Significance was determined by a 2-tailed student's t-test.

G and H). Data from (D-F) was combined to show the trends in LPL folding over time for reactions using HEK293 or HEK293∆LMF1 cells. Each point is the average of three independent experiments, and error bars represent the standard deviation.
Lipase Maturation Factor 1 Affects  Redox Homeostasis in the Endoplasmic Reticulum (2018)
Figure 4

Figure 4: LMF1 contributes to redox homeostasis in the ER.

A, B). Differential sensitivity of HEK293 cells containing and lacking LMF1 (A) and cld/cld and wt/cld MEFs (B) to drugs that perturb cellular redox homeostasis. Cells were treated with BSO, DTT, or tunicamycin as described in materials and methods. Treated cell and untreated cells were counted, and the fraction of surviving cells was calculated. Three wells were counted and averaged for each data point, and three independent trials were carried out per condition. Significance was determined by a 2-tailed student's t-test.

C). The redox sensor ERroGFP-S4 shows that HEK293∆LMF1 cells have a more oxidized ER than HEK293 and HEK293+LMF1 cells both untreated and when stressed by expression of LPL-mCherry. A two-tailed student's t-test was used to calculate the significance of the difference in the ratio of oxidized/reduced GFP fluorescence intensity between cell types. Each data point is one of three spots measured per cell, 17 cells were measured per condition. Significance was determined by a 2-tailed student's t-test.
Lipase Maturation Factor 1 Affects  Redox Homeostasis in the Endoplasmic Reticulum (2018)
Figure 5

Figure 5: Conserved cysteines in LMF1 are important to LPL secretion

A). The LMF1 protein sequence from multiple species was aligned using Clustal Omega (Sievers et al., 2011)

B). Schematic of the membrane topology of LMF1 showing the location of all the cysteines.

C). Secretion of LPL-V5 from HEK293∆LMF1 cells transiently transfected with LMF1 bearing the indicated cysteine to alanine mutations. Lipase levels in the lysate and pellet, and LMF1 levels in the pellet (α-HIS), are also probed. GAPDH serves as a loading control.
Lipase Maturation Factor 1 Affects  Redox Homeostasis in the Endoplasmic Reticulum (2018)
Figure 6

Figure 6: Analysis of LMF1's redox-dependent interactions.

A) α-His Western blot to detect the redox state of LMF1. In the first two lanes samples were untreated and resolved in reducing or non-reducing loading dye. The next 4 lanes were subject to the indicated treatments prior to cell lysis then treated with AMS prior to non-reducing SDS-PAGE.

B) α-His Western blot to detect acid trapped partners of LMF1. Samples were TCA precipitated, treated with NEM, and LMF1 was purified via its C-terminal His tag. Samples were resolved by reducing or nonreducing SDS-PAGE. The lanes marked -LMF1 are HEK293∆LMF1 cells included as a negative control.

C) Samples were prepared as in (B), but were probed with an antibody for TRX.
Lipase Maturation Factor 1 Affects  Redox Homeostasis in the Endoplasmic Reticulum (2018)
Figure 7

Figure 7: Fibronectin and LDLR depend on LMF1 for secretion.

A). Bottom, center and top slices from a z-stack of a cld/wt or cld/cld MEF cell. YPet-tagged fibronectin is in green and mCherry-Sec61β, an ER marker, is in red. Note fibronectin assembly into the extracellular matrix in cld/wt cells.

B). cld/wt cells had significantly higher amounts of fibronectin outside of the ER than cld/cld cells by a two-tailed student's t-test.

C). Bottom, center and top slices show that LDLR is retained in the ER in cells HEK293∆LMF1 cells. LDLR-GFP is in green and mCherry-Sec61β is in red. Note the clumping of LDLR in the ER in HEK293∆LMF1 cells.

D). The ratio of LDLR within the ER/total LDLR was significantly higher for HEK293∆LMF1 cells than HEK293 or HEK293+LMF1 cells by a 2-tailed student's t-test.

Data information: For all panels >10 cells were measured per genotype. All scale bars are 10 µm.
Co-inhibition of immunoproteasome subunits LMP2 and LMP7 is required to block autoimmunity (2018)
Figure 1

Figure 1. PRN1126 is selective for the chymotrypsin-like activity of the immunoproteasome.

A Structure of PRN1126, a reversible covalent selective LMP7 inhibitor.

B, C Hydrolysis of fluorogenic substrates (Suc-LLVY-AMC) for the chymotrypsin-like activity of human (B) or mouse (C) 20S constitutive proteasome (CP) or 20S immunoproteasome (IP) at various PRN1126 concentrations. Data are presented as the means ± s.d. from quadruplicate assays. The highest fluorescence value was set to 100%. The experiments were repeated twice with similar results.
Co-inhibition of immunoproteasome subunits LMP2 and LMP7 is required to block autoimmunity (2018)
Figure 2

Figure 2. PRN1126 does not affect dextrane sulfate sodium (DSS)-induced colitis or experimental autoimmune encephalomyelitis (EAE).

A Presentation of UTY246-254 on splenocytes after exposure to indicated concentrations of PRN1126. Data are presented as the mean absorbance ± s.d. of three replicate cultures. The experiment has been performed twice, yielding similar results.

B Flow cytometry analysis of H-2Kb surface expression on splenocytes derived from C57BL/6 mice treated with the indicated concentrations of PRN1126 overnight. Pooled data from three independent experiments (n=9) are shown as the means of median fluorescent intensity ± SEM. All data were statistically compared to the DMSO treated group. ***P < 0.001. One-way ANOVA.

C, D Splenocytes from C57BL/6 mice (C) or human PBMCs (D) were exposed (continuous treatment) to 300 nM PRN1126 or vehicle (DMSO) or 300 nM ONX 0914 and stimulated with LPS overnight. IL-6 concentrations in the supernatant were analyzed by ELISA. (C) IL-6 concentrations are presented as mean and individual data points from triplicate wells. The experiment has been performed twice, yielding similar results. (D) Data are presented as single dots from three independent donors. The highest cytokine concentration was set to 100%. *P < 0.05. Unpaired student´s t-test.

E Colitis was induced by oral administration of 3 % DSS for 5 d. Mice were treated daily (s.c.) with either PRN1126 (40 mg/kg), or ONX 0914 (10 mg/kg), or vehicle. Data points represent mean ± SEM of 15 mice pooled from three independent experiments. All data were statistically compared to the vehicle treated group. *P < 0.05, ***P < 0.001. Two-way ANOVA.

F Mice were immunized with MOG35-55 peptide and were monitored daily for clinical symptoms of EAE. Mice were treated three times a week (s.c.) with either PRN1126 (40 mg/kg), or ONX 0914 (10 mg/kg), or vehicle. All data were statistically compared to the vehicle treated group. *P < 0.05, **P < 0.01, ***P < 0.001. Two-way ANOVA. Shown are the means of the clinical scores ± SEM (n = 6 per group). The experiments have been performed twice, yielding similar results.
Co-inhibition of immunoproteasome subunits LMP2 and LMP7 is required to block autoimmunity (2018)
Figure 3

Figure 3. ONX 0914 but not PRN1126 inhibits LMP2.

A Hydrolysis of fluorogenic substrates (Ac-PAL-AMC) for LMP2-activity of human (upper panel) or mouse (lower panel) 20S immunoproteasome at various concentrations of PRN1126 or ONX 0914. Data are presented as the means of fluorescence ± s.d. from quadruplicate assays. The experiments were repeated three times with similar results.

B Altered electrophoretic mobility of IP subunits by covalent modification with ONX 0914. Ficoll enriched lymphocytes from C57BL/6 mice were treated with DMSO or 300 nM ONX 0914 for 2 h in vitro. SDS-PAGE and immunoblotting against indicated proteins was performed. Shown are representative western blots out of three independent experiments with similar outcome.
Co-inhibition of immunoproteasome subunits LMP2 and LMP7 is required to block autoimmunity (2018)
Figure 4

Figure 4. LMP7 and LMP2 co-inhibition reduces H-2Kb surface expression on splenocytes.

Flow cytometry analysis of H-2Kb surface expression on splenocytes derived from C57BL/6 wild type (left side) or LMP7-/- (right side) mice treated with vehicle (DMSO), ONX 0914 (300 nM), PRN1126 (300 nM), ML604440 (300 nM), LU-001i (300 nM), PRN1126+ML604440 (300 nM each), or PRN1126+LU-001i (300 nM each) overnight. Unstained splenocytes were used as negative control. Pooled data from three independent experiments (n=9) are shown as the means of median fluorescent intensity ± SEM. All data were statistically compared to the DMSO treated group. **P < 0.01, ***P < 0.001. One-way ANOVA.
Co-inhibition of immunoproteasome subunits LMP2 and LMP7 is required to block autoimmunity (2018)
Figure 5

Figure 5. The reduction of IL-6 production and the suppression of Th17 differentiation relies on the co-inhibition of LMP7 and LMP2.

A, B Splenocytes from C57BL/6 mice (A) or human PBMCs (B) were incubated (continuous treatment) with DMSO, ONX 0914 (300 nM), PRN1126 (300 nM), ML604440 (300 nM), LU-001i (300 nM), PRN1126+ML604440 (300 nM each), or PRN1126+LU-001i (300 nM each) and stimulated with LPS overnight. The IL-6 concentration in the supernatant was analyzed by ELISA. The highest IL-6 concentration from each experiment was set to 100%. (A) Data are presented as the mean ± SEM of 4 independent experiments each measured in triplicates. (B) Data are presented as the mean ± SEM of 6 different healthy donors. All data were statistically compared to the DMSO treated group. ***P < 0.001. One-way ANOVA.

C The differentiation of CD4+ T cells isolated from the spleens of C57BL/6 mice that were exposed (continuous treatment) to DMSO, ONX 0914 (300 nM), PRN1126 (300 nM), ML604440 (300 nM), LU-001i (300 nM), PRN1126+ML604440 (300 nM each), or PRN1126+ LU-001i (300 nM each) and that were stimulated with plate bound antibodies to CD3/CD28 in the presence of TGF-β and IL-6 and scavenging antibodies neutralising IL-4 and IFN-γ was measured in 3 days cultures. IL-17 expression was detected by intracellular cytokine staining after a short restimulation with PMA/Ionomycin. IL-17 expression in activated T cells cultured in the absence of Th17-polarizing conditions is shown as a comparison (no cocktail). Values reflect the percentage of CD4+ cells that are IL-17A+. Data are presented as the means ± SEM of 3 independent experiments each measured in triplicates. All data were statistically compared to the DMSO treated group. ***P < 0.001. One-way ANOVA.
TEX11 is mutated in infertile men with azoospermia and regulates genome-wide recombination rates in mouse. (2015)
Figure 2

A wild-type <italic>Tex11</italic> knockin allele at the autosomal <italic>Tex19.1</italic> locus rescues male sterility in <italic>Tex11</italic>-null adult mice

Schematic diagram of Tex11 gene structure, Tex19.1 gene structure, targeting vector and the final Tex11 knockin allele containing the wild-type Tex11 ORF.

Western blot analysis of TEX11 in the testes of 3-month-old mice with different Tex11 gene dosages. ACTB serves as a loading control.

Testis weight of 3-month-old males.

Sperm count of 3-month-old males.

Histological analysis of testes from adult wild-type, Tex11 KI/KO, and Tex11−/Y males. RS, round spermatids; ES, elongating spermatids; Pa, pachytene spermatocytes. Scale bar, 50 μm.

The Tex11 knockin allele rescues chromosomal synapsis defects in Tex11−/Y spermatocytes in 3-month-old males. Chromosomal synapsis defects were assessed by SYCP1 and SYCP2 immunostaining of spread nuclei from 100 pachytene spermatocytes per male; for each genotype, three males were analyzed. Abbreviations for Tex11 genotypes: −/Y, Tex11 knockout (Yang et al, 2008); KI;−/Y, Tex11 knockin and knockout; +/Y, wild type; KI;+/Y, Tex11 knockin plus wild-type Tex11.

Data information: All statistical analyses were performed using Student’s t-test. n.s.: not statistically significant.
TEX11 is mutated in infertile men with azoospermia and regulates genome-wide recombination rates in mouse. (2015)
Figure 3

Defective meiotic progression in the first wave of spermatogenesis in <italic>Tex11</italic> KI/KO males

The knockin allele expresses the wild-type Tex11 ORF.

Reduced testis weight in juvenile but not adult Tex11 KI; −/Y males. The time frames of the first wave and adult waves of spermatogenesis are indicated.

Increased chromosomal asynapsis in spermatocytes and reduced levels of TEX11 protein in the testes of 25-day-old Tex11 KI/KO males. Chromosomal synapsis defects were assessed by SYCP1 and SYCP2 immunostaining of spread nuclei from 100 pachytene spermatocytes per male; for each genotype, three males were analyzed.

Pachytene spermatocytes from 25-day-old Tex11 KI/KO males contain significantly fewer MLH1 foci compared to wild-type spermatocytes. Values are shown as average ± standard deviation. Error bar is standard deviation.

Data information: Statistical analyses were performed with Student’s t-test.
TEX11 is mutated in infertile men with azoospermia and regulates genome-wide recombination rates in mouse. (2015)
Figure 4

<italic>Tex11</italic> dosage affects the number of MLH1 foci in meiotic germ cells in both sexes

The number of MLH1 foci in pachytene spermatocytes from 3-month-old males positively correlates with increasing Tex11 gene dosage.

Quantification of MLH1 foci in pachytene oocytes from the ovaries of Tex11−/−, Tex11+/−, and wild-type embryonic day-17.5 (E17.5) fetuses reveals a positive correlation of Tex11 gene dosage and the number of MLH1 foci.

Data information: Statistical analyses were performed with Student’s t-test.
TEX11 is mutated in infertile men with azoospermia and regulates genome-wide recombination rates in mouse. (2015)
Figure 6

A single amino acid change (V749A) in TEX11 causes severe defects in chromosomal synapsis in mice

Data shown are from 3-month-old Tex11 KI/KO males with wild-type or mutant knockin allele (n, number of males analyzed per genotype) except for mating test (E).

A Western blot analysis reveals comparable levels of TEX11 protein in the testes of wild-type and point mutant KI/KO males. ACTB serves as a control.

B–D Body weight (B), testis weight (C), and significantly reduced sperm count (per pair of epididymides) in Tex11 KI(V749A)/KO males (D).

E Mating test of 3- to 5-month-old males.

F Dramatically increased chromosomal asynapsis in spermatocytes from Tex11 KI(V749A)/KO males. 100 pachytene spermatocytes per mouse were examined by surface spread analysis.

G Number of MLH1 foci in pachytene spermatocytes. Note a significantly higher number of MLH1 foci in spermatocytes from KI(Q173R)/KO males.

Data information: Values are shown as average ± standard deviation. n.s.: not statistically significant.
The DNA Damage Response acts as a safeguard against harmful DNA-RNA hybrids of different origins (2019)
Figure 1

Figure 1. Screening for DDR factors involved in R loop homeostasis. (A) Top hit selected candidates are listed. The plot shows relative S9.6 nuclear intensity values for cells transformed with each of the indicated pool of siRNAs. (B) Representative images of HeLa cells immunostained with S9.6 and nucleolin antibodies after transfection with at least a pool of two siRNAs of each original pool. (C) Relative S9.6 signal intensity per nucleus after nucleolus signal removal in HeLa cells transfected with the indicated siRNAs. More than 250 total cells from three independent experiments were considered. The median of each population is shown. Boxes and whiskers indicate 25-75 and 10-90 percentiles, respectively. ***p<0.001 (Mann-Whitney U test).
The DNA Damage Response acts as a safeguard against harmful DNA-RNA hybrids of different origins (2019)
Figure 2

Figure 2. R loop accumulation after depletion of the selected DNA-Damage Checkpoint (DDC) and Postreplicative Repair (PRR) candidates. (A) Relative DRIP-qPCR signal values at RPL13A, APOE, MIB2 and RHOT2 genes in HeLa cells transfected with the indicated siRNAs and treated in vitro with RNase H pre-immunoprecipitation where indicated. The mean ± SEM from at least three independent experiments is shown. *p<0.05, **p<0.01, ***p<0.001 (one-tailed paired t-test). (B) Representative images of immunostaining with S9.6 and anti-nucleolin antibodies in HeLa cells transfected with the indicated siRNAs (C) Relative S9.6 signal intensity per nucleus after nucleolus signal removal in HeLa cells after cytoplasm pre-extraction (CE) and treated in vitro with RNaseIII and RNase H where indicated. More than 500 total cells from three independent experiments were considered. The median of each population is shown. Boxes and whiskers indicate 25-75 and 10-90 percentiles, respectively. **p<0.01, ***p<0.001 (Mann-Whitney U test).

Black stars denote significant increases whereas red stars denote significant decreases.