Journal: Autophagy

Article Title: Defective recognition of LC3B by mutant SQSTM1/p62 implicates impairment of autophagy as a pathogenic mechanism in ALS-FTLD

doi: 10.1080/15548627.2016.1170257

Figure Lengend Snippet: ALS-FTLD-associated SQSTM1 mutations impact on the recognition of LC3B (SQSTM1 L341V ) or ubiquitin (SQSTM1 G425R ) in vitro. Mutations as indicated (or wild type, WT) were introduced into the full-length GST-SQSTM1 sequence and affinity isolation assays (LC3B and ubiquitin on beads) were performed at 37°C. Bacterial lysates containing the GST-SQSTM1 fusions were incubated with glutathione- (G), control- (C), LC3B (LC3), and ubiquitin-Sepharose (Ub) beads and captured proteins were detected by western blotting (anti-SQSTM1 antibodies). A representative blot is shown; see Fig. S1 for quantification of 3 independent experiments.

Article Snippet: One ml of each diluted lysate was incubated at 37°C with excess glutathione-Sepharose (GE Healthcare, 17-0756-01), ubiquitin-Sepharose (10 mg/ml human ubiquitin immobilized on CNBr-activated Sepharose 4B [Sigma-Aldrich, C9142]), LC3B-Sepharose (1 mg/ml LC3B immobilized on CNBr-activated Sepharose 4B) or control-Sepharose (CNBr-activated Sepharose prepared without ubiquitin or LC3B).

Techniques: In Vitro, Sequencing, Isolation, Incubation, Western Blot

Journal: Autophagy

Article Title: Defective recognition of LC3B by mutant SQSTM1/p62 implicates impairment of autophagy as a pathogenic mechanism in ALS-FTLD

doi: 10.1080/15548627.2016.1170257

Figure Lengend Snippet: ESI-MS indicates weaker binding of the LIR (L341V) to LC3B compared to WT LIR. (A) Native ESI-MS spectrum of an equimolar mixture of WT LIR and LIR (L341V) peptides (5 µM, residues 332 to 351). (B) LIR peptide mixture titrated with 5 µM LC3B. Top, full spectrum indicating free LIR (gray filled circle, mixture of WT LIR and LIR [L341V]), free LC3B and LC3B-LIR complexes of indicated (multiple) charge states. Below, zoomed-in spectra showing m/z values of the free and bound LIR complexes at the indicated charge states.

Article Snippet: One ml of each diluted lysate was incubated at 37°C with excess glutathione-Sepharose (GE Healthcare, 17-0756-01), ubiquitin-Sepharose (10 mg/ml human ubiquitin immobilized on CNBr-activated Sepharose 4B [Sigma-Aldrich, C9142]), LC3B-Sepharose (1 mg/ml LC3B immobilized on CNBr-activated Sepharose 4B) or control-Sepharose (CNBr-activated Sepharose prepared without ubiquitin or LC3B).

Techniques: Binding Assay

Journal: Autophagy

Article Title: Defective recognition of LC3B by mutant SQSTM1/p62 implicates impairment of autophagy as a pathogenic mechanism in ALS-FTLD

doi: 10.1080/15548627.2016.1170257

Figure Lengend Snippet: Isothermal titration calorimetry (ITC) binding isotherms from titrating LC3B with LIR peptides at 25°C. Binding isotherms were fitted to a one-site binding model and K d values determined. Starting concentration of the LC3B was approximately 30 µM, and of LIR peptide (residues 332 to 351) stocks approximately 350 µM. Black diamond, LIR (L341V); gray circle WT LIR.

Article Snippet: One ml of each diluted lysate was incubated at 37°C with excess glutathione-Sepharose (GE Healthcare, 17-0756-01), ubiquitin-Sepharose (10 mg/ml human ubiquitin immobilized on CNBr-activated Sepharose 4B [Sigma-Aldrich, C9142]), LC3B-Sepharose (1 mg/ml LC3B immobilized on CNBr-activated Sepharose 4B) or control-Sepharose (CNBr-activated Sepharose prepared without ubiquitin or LC3B).

Techniques: Isothermal Titration Calorimetry, Binding Assay, Concentration Assay

Journal: Autophagy

Article Title: Defective recognition of LC3B by mutant SQSTM1/p62 implicates impairment of autophagy as a pathogenic mechanism in ALS-FTLD

doi: 10.1080/15548627.2016.1170257

Figure Lengend Snippet: NMR titrations indicate selective perturbations of LC3B residues upon binding of LIR peptides. (A) 1 H- 15 N-HSQC spectrum of LC3B (0.25 mM dark gray) overlaid with the spectrum of the complex of LC3B with WT LIR (0.5 mM blue, residues 332 to 351) (ratio of 1:2) showing extensive chemical shift perturbations (CSPs) across the spectrum induced by ligand binding at 298 K; (B) Expansion of the region highlighted in (A) showing overlap between the spectrum of LC3B (dark gray), LC3B+WT LIR (blue) and LC3B + LIR (L341V) (green) illustrating a number of key residues within the LIR binding pocket that are perturbed to different extents by the WT LIR and LIR (L341V). Arrows identify the shifts of V33, F52 and V54 in the 2 complexes, while the majority of other residues show only small differences between the spectra of the 2 complexes (overlap of blue and green peaks), demonstrating selective effects on residues in direct contact with the LIRs.

Article Snippet: One ml of each diluted lysate was incubated at 37°C with excess glutathione-Sepharose (GE Healthcare, 17-0756-01), ubiquitin-Sepharose (10 mg/ml human ubiquitin immobilized on CNBr-activated Sepharose 4B [Sigma-Aldrich, C9142]), LC3B-Sepharose (1 mg/ml LC3B immobilized on CNBr-activated Sepharose 4B) or control-Sepharose (CNBr-activated Sepharose prepared without ubiquitin or LC3B).

Techniques: Binding Assay, Ligand Binding Assay

Journal: Autophagy

Article Title: Defective recognition of LC3B by mutant SQSTM1/p62 implicates impairment of autophagy as a pathogenic mechanism in ALS-FTLD

doi: 10.1080/15548627.2016.1170257

Figure Lengend Snippet: Chemical shift mapping of the binding of the WT LIR and LIR (L341V) peptides (residues 332 to 351) to 15 N-LC3B. (A) Chemical shift mapping of the WT LIR. Weighted chemical shift pertubation (CSP) data showing the residues of 15 N-LC3B (0.25 mM) that are perturbed upon WT LIR binding at a final ratio of 1:2 (LC3B:LIR) at 298 K. All CSPs above 1.0 are indicated. (B) Difference in CSP effects between WT LIR and LIR (L341V) sequences from NMR titrations at a final ratio of 1:2 (LC3B:LIR) at 298 K. The indicated residues showed CSPs that are substantially different between the 2 complexes (greater in the WT LIR). (C) Representation of binding surface of LC3B with WT LIR, generated by highlighting residues determined from NMR chemical shift mapping experiments. LC3B residues in blue showed the greatest CSP values. Backbone representation of the LIR peptide also indicated (residues DDDWTHLS, 335 to 342 shown). (D) Residues showing substantially different CSPs from the NMR titrations of LC3B with WT LIR compared to LIR (L341V), highlighted on the LC3B structure. Note the close correlation with the binding cleft in proximity to L341 of the LIR peptide (site of L341V indicated).

Article Snippet: One ml of each diluted lysate was incubated at 37°C with excess glutathione-Sepharose (GE Healthcare, 17-0756-01), ubiquitin-Sepharose (10 mg/ml human ubiquitin immobilized on CNBr-activated Sepharose 4B [Sigma-Aldrich, C9142]), LC3B-Sepharose (1 mg/ml LC3B immobilized on CNBr-activated Sepharose 4B) or control-Sepharose (CNBr-activated Sepharose prepared without ubiquitin or LC3B).

Techniques: Binding Assay, Generated

Journal: Science Advances

Article Title: Discovery of pan-VEGF inhibitory peptides directed to the extracellular ligand-binding domains of the VEGF receptors

doi: 10.1126/sciadv.1600611

Figure Lengend Snippet: ( A ) The extracellular domain of mouse VEGFR-3 was immobilized on microtiter wells and incubated with the X6 phage display library. Bar graph shows enrichment in the number of phage recovered [in transducing units (TU)] after consecutive rounds of selection (I, II, and III). (*) Round I was not quantified to prevent the loss of phage displaying unique peptides. ( B ) Peptide identified by sequencing phage bound to VEGFR-3 (round III) ( n , number of phages sequenced). ( C and D ) Binding of control phage Fd (white bars) and phage PCAIWF (B, black bars) and WVCSGG (C, black bars) to VEGF receptors and co-receptors immobilized on microtiter wells. ( E and F ) Inhibition of phage PCAIWF (E) or WVCSGG (F) binding to immobilized VEGFR-3 by synthetic peptide PCAIWF or control peptide (CARAC). The minus sign indicates that no synthetic peptide was added to the assay. ( G ) Dose-response assay. Phage PCAIWF was incubated with immobilized VEGFR-3 in the presence of synthetic peptides PCAIWF, PSAIWF, or CARAC (control). Percentage relative to phage binding in the absence of competing peptide. In all cases, bars represent means ± SEM from triplicate plating. Statistics, Student’s t test (** P ≤ 0.01 and *** P ≤ 0.001).

Article Snippet: Antibodies and other reagents were obtained commercially: anti-human VEGF (AF-293-NA), anti-human VEGF-C (AF752), anti-human PlGF (AF-264-PB), anti-human PDGF-BB (AF-220-NA), anti-human FGF-basic (AF-233-NA), anti-mouse/rat NRP-1 (AF566), and anti-mouse/rat NRP-2 (AF567) were from R&D Systems; anti-fd Bacteriophage-Biotin Conjugate (B2661) was from Sigma-Aldrich; secondary antibodies IRDye 680LT Donkey anti-goat IgG and IRDye 680LT Streptavidin were from LI-COR.

Techniques: Incubation, Selection, Sequencing, Binding Assay, Control, Inhibition

Journal: Science Advances

Article Title: Discovery of pan-VEGF inhibitory peptides directed to the extracellular ligand-binding domains of the VEGF receptors

doi: 10.1126/sciadv.1600611

Figure Lengend Snippet: ( A ) Binding of phage PCAIWF to immobilized VEGFR-3 in the presence or absence of VEGF-A or VEGF-C (10 ng/ml). ( B ) Binding of phage PCAIWF to immobilized VEGFR-3 in the presence of increasing concentrations of VEGF-C. Percentage relative to phage binding in the absence of VEGF-C. ( C ) Cartoon showing the three-dimensional structure of the complex VEGF-C (red) bound to VEGFR-2 IgD2-3 (shown in orange and green, respectively) (Protein Data Bank #2X1W). ( D ) Analysis by SDS–polyacrylamide gel electrophoresis of purified recombinant IgD2 and IgD2-3 proteins containing the ligand-binding domain of VEGFR-3. ( E ) Binding of phage PCAIWF to VEGFR-3 and its recombinant Ig domains immobilized on microtiter wells in the presence or absence of the synthetic peptide PCAIWF or its scramble version, IFCAPW (100 μg/ml). Phage binding was quantified by FLISA using an anti-bacteriophage sera. ( F ) Binding of VEGF-C to microtiter wells coated with immobilized recombinant ligand binding domains IgD2 and IgD2-3 of VEGFR-3 in the presence or absence of synthetic peptides PCAIWF and WVCSGG or the scramble control peptide (IFCAPW). For phage experiments (A and B), bars represent mean ± SEM from triplicate plating; for FLISA assays ( E to G ), bars represent means ± SEM from duplicate wells. Statistics, Student’s t test [not significant (N.S.), P > 0.05; * P ≤ 0.05 and *** P ≤ 0.001].

Article Snippet: Antibodies and other reagents were obtained commercially: anti-human VEGF (AF-293-NA), anti-human VEGF-C (AF752), anti-human PlGF (AF-264-PB), anti-human PDGF-BB (AF-220-NA), anti-human FGF-basic (AF-233-NA), anti-mouse/rat NRP-1 (AF566), and anti-mouse/rat NRP-2 (AF567) were from R&D Systems; anti-fd Bacteriophage-Biotin Conjugate (B2661) was from Sigma-Aldrich; secondary antibodies IRDye 680LT Donkey anti-goat IgG and IRDye 680LT Streptavidin were from LI-COR.

Techniques: Binding Assay, Polyacrylamide Gel Electrophoresis, Purification, Recombinant, Ligand Binding Assay, Fluorophore-linked Immunoabsorbent Assay, Control

Journal: Science Advances

Article Title: Discovery of pan-VEGF inhibitory peptides directed to the extracellular ligand-binding domains of the VEGF receptors

doi: 10.1126/sciadv.1600611

Figure Lengend Snippet: ( A ) Representation of the VEGF family, their receptors, and pattern of interaction. ( B to F ) Recombinant proteins for the human VEGFR-3 (B), VEGFR-2 (C and E), and VEGFR-1 (D and F) extracellular domains were immobilized on microtiter wells and incubated with the human ligands VEGF-C (B and C), PlGF (D), and VEGF-A (E and F) in the presence or absence of synthetic peptides PCAIWF and PSAIWF or the scramble control peptide (IFCAPW). Growth factors bound to the wells were quantified by FLISA using immunospecific antibodies and fluorescent detection. Bars represent means ± SEM from duplicate wells. Statistics, analysis of variance (ANOVA) (Tukey’s multiple comparison test) (* P ≤ 0.05; ** P ≤ 0.01 and *** P ≤ 0.001).

Article Snippet: Antibodies and other reagents were obtained commercially: anti-human VEGF (AF-293-NA), anti-human VEGF-C (AF752), anti-human PlGF (AF-264-PB), anti-human PDGF-BB (AF-220-NA), anti-human FGF-basic (AF-233-NA), anti-mouse/rat NRP-1 (AF566), and anti-mouse/rat NRP-2 (AF567) were from R&D Systems; anti-fd Bacteriophage-Biotin Conjugate (B2661) was from Sigma-Aldrich; secondary antibodies IRDye 680LT Donkey anti-goat IgG and IRDye 680LT Streptavidin were from LI-COR.

Techniques: Recombinant, Incubation, Control, Fluorophore-linked Immunoabsorbent Assay, Comparison

Journal: Science Advances

Article Title: Discovery of pan-VEGF inhibitory peptides directed to the extracellular ligand-binding domains of the VEGF receptors

doi: 10.1126/sciadv.1600611

Figure Lengend Snippet: ( A ) Immunoblot analysis of phosphorylated and total forms of ERK1/2 in LECs incubated with VEGF-A, VEGF-C, or FGF (100 ng/ml) in the presence or absence of peptide PCAIWF or scramble (IFCAPW) (30 μg/ml). ( B ) Ratio of fluorescent intensity for phosphorylated and total ERK1/2. Bars represent means ± SEM from three independent measurements of the immunoblot membrane. Two independent experiments were performed with similar results. Bars represent means ± SEM from triplicate readings. Statistics, ANOVA (Tukey’s multiple comparison test) (* P ≤ 0.05).

Article Snippet: Antibodies and other reagents were obtained commercially: anti-human VEGF (AF-293-NA), anti-human VEGF-C (AF752), anti-human PlGF (AF-264-PB), anti-human PDGF-BB (AF-220-NA), anti-human FGF-basic (AF-233-NA), anti-mouse/rat NRP-1 (AF566), and anti-mouse/rat NRP-2 (AF567) were from R&D Systems; anti-fd Bacteriophage-Biotin Conjugate (B2661) was from Sigma-Aldrich; secondary antibodies IRDye 680LT Donkey anti-goat IgG and IRDye 680LT Streptavidin were from LI-COR.

Techniques: Western Blot, Incubation, Membrane, Comparison

Journal: Science Advances

Article Title: Discovery of pan-VEGF inhibitory peptides directed to the extracellular ligand-binding domains of the VEGF receptors

doi: 10.1126/sciadv.1600611

Figure Lengend Snippet: ( A ) Tube formation by HUVECs in Matrigel induced by VEGF or VEGF-C in the presence or absence of peptide PCAIWF or scramble (500 μg/ml, embedded in the Matrigel layer). ( B ) Number of tubes formed between endothelial cells. Bars represent means ± SEM from triplicate wells. Statistics, Student’s t test (* P ≤ 0.05). Two independent experiments were performed with similar results.

Article Snippet: Antibodies and other reagents were obtained commercially: anti-human VEGF (AF-293-NA), anti-human VEGF-C (AF752), anti-human PlGF (AF-264-PB), anti-human PDGF-BB (AF-220-NA), anti-human FGF-basic (AF-233-NA), anti-mouse/rat NRP-1 (AF566), and anti-mouse/rat NRP-2 (AF567) were from R&D Systems; anti-fd Bacteriophage-Biotin Conjugate (B2661) was from Sigma-Aldrich; secondary antibodies IRDye 680LT Donkey anti-goat IgG and IRDye 680LT Streptavidin were from LI-COR.

Techniques:

Journal: Nature Communications

Article Title: Phase separation of Nur77 mediates celastrol-induced mitophagy by promoting the liquidity of p62/SQSTM1 condensates

doi: 10.1038/s41467-021-26295-8

Figure Lengend Snippet: a Representative image of celastrol-induced mitophagy in HeLa, Nur77 −/− HeLa, and Nur77 −/− HeLa cells transfected with Myc-Nur77 by EGFP-mCherry-COX8 assay as described in Methods. Scale bar, 10 μm. b Representative images of celastrol-induced mitophagy in MEFs and p62 −/− MEFs by EGFP-mCherry-COX8 assay as described in Methods. Scale bar, 10 μm. c Colocalization of Nur77, LC3, and p62 with mitochondria within mitophagosome/autolysosome. Upper panel: Electron micrographs of HeLa cells stained with 15 nm immunogold-conjugated Nur77 antibody to detect Nur77 (red), and 10 nm immunogold-conjugated p62 antibody to detect p62 (green). Bottom panel: Electron micrographs of HeLa cells stained with 15 nm immunogold-conjugated LC3 antibody to detect LC3, and 10 nm immunogold-conjugated p62 antibody to detect p62. Cells were treated for 1 h with celastrol. The blue dotted line indicates mitophagosome/autolysosome. Mito mitochondrion, Scale bar, 200 nm. d Representative images showing Hsp60, a mitochondrial marker, in the liver tissue from wild-type and Nur77 −/− mice in aging model. Young mice, 8 weeks old. Aged mice, 2 years old. Scale bar, 10 μm. e Statistical analysis of mitochondrial size was represented from liver tissue. Left graph, n = 316, 253, 267, and 287, respectively; Right graph, n = 3 biologically independent samples. A two-tailed unpaired Student’s t -test was used for statistical analysis, and data were presented as mean values ± SEM. f The expression of Nur77 protein in the liver tissue from wild-type and Nur77 −/− mice in the aging model. g Representative images of EGFP-mCherry-COX8 in the liver from wild-type or Nur77 −/− mice in the aging model. Purple arrows indicate mitophagy. Scale bar, 2 μm. h Quantification of cells showing mCherry-COX8 accumulation on liver tissue. Two-tailed unpaired Student’s t -test was used for statistical analysis, and data were presented as mean values ± SEM ( n = 5 mice per group). Data represent at least three independent experiments. Source data are provided as a Source Data file.

Article Snippet: For immunostaining, the following antibodies were used: Nur77 for HeLa cell study (CST, 3960S, dilution: 1:100), Nur77 (M-210) (Santa Cruz, sc-5569, dilution: 1:50), Nur77 for mouse study (Affinity, DF7850, dilution:1:200), CD68 (Abcam, AB955, dilution: 1:200), SQSTM1/p62 (Abcam, AB56416, dilution: 1:400), Myc (9E10) (Santa Cruz, sc-40, dilution: 1:100), FLAG (Sigma-Aldrich, F1804, dilution: 1:400), HA (Santa Cruz, sc-7392, dilution: 1:100), LC3 (MBL, PM036, dilution: 1:200), Hsp60 (Santa Cruz, sc-13115, dilution: 1:100), FIP200 (CST, 12436, dilution: 1:100), ATG16L1 (Proteintech, 19812, dilution: 1:100), WIPI2 (Proteintech, 15432, dilution: 1:100), ULK1 (Santa Cruz, Sc-390904, dilution: 1:50), MitoTracker Red FM (Thermo Fisher Scientific, M22425, dilution: 1:20000), MitoTracker Deep Red FM (Thermo Fisher Scientific, M22426, dilution: 1:20000), LysoTracker Deep Red FM (Thermo Fisher Scientific, L12492, dilution: 1:20,000), Goat anti-Rabbit (A10523) and anti-Mouse (A10524) IgG (H+L) Cross-Adsorbed Secondary Antibody, Cy5 (Thermo Fisher Scientific, dilution: 1:200), Cy3-AffiniPure Goat Anti-Rabbit (111-165-003) and Anti-Mouse (115-165-003) IgG (H+L) (Jackson, dilution: 1:200), FITC-AffiniPure Rabbit Anti-Goat IgG (H+L) FITC (Yeasen, 33707ES60, dilution: 1:200).

Techniques: Transfection, Staining, Marker, Two Tailed Test, Expressing

Journal: Nature Communications

Article Title: Phase separation of Nur77 mediates celastrol-induced mitophagy by promoting the liquidity of p62/SQSTM1 condensates

doi: 10.1038/s41467-021-26295-8

Figure Lengend Snippet: a Representative images showing the time-dependent effect on celastrol induction of cytoplasmic Nur77 body formation. Bottom panels: quantitative analysis of the number and size of Nur77/p62 body formation. Bottom left graph, n = 4 biologically independent samples; Bottom right graph, n = 20, 23, 25, and 19, respectively. Data were presented as mean values ± SEM. Scale bar, 10 μm. b Real-time images showing the formation and fusion of GFP-Nur77 and mCherry-p62 droplets in HeLa cells after treatment with celastrol (2 μM) for 1 h. White arrows indicate droplets formation and fusion (see also Supplementary Movie ). Scale bar, 10 μm. c Representative images illustrating the role of celastrol in promoting p62 body formation in a Nur77-dependent manner immunostaining. Nur77 −/− HeLa cells were also transfected with GFP-Nur77 to determine its effect on p62 body formation. The diameter of the biggest p62 puncta in each cell was measured. The number of p62 puncta >0.5 μm in each cell was assessed. A two-tailed unpaired Student’s t -test was used for statistical analysis, and data are presented as mean values ± SEM ( n = 5 biologically independent samples). d FRAP analysis of the effect of Nur77 in regulating p62 mobility in HeLa cells. Data were presented as means ± SEM ( n = 3 independent experiments). Scale bar, 1.5 μm. Data represent at least three independent experiments. Source data are provided as a Source Data file.

Article Snippet: For immunostaining, the following antibodies were used: Nur77 for HeLa cell study (CST, 3960S, dilution: 1:100), Nur77 (M-210) (Santa Cruz, sc-5569, dilution: 1:50), Nur77 for mouse study (Affinity, DF7850, dilution:1:200), CD68 (Abcam, AB955, dilution: 1:200), SQSTM1/p62 (Abcam, AB56416, dilution: 1:400), Myc (9E10) (Santa Cruz, sc-40, dilution: 1:100), FLAG (Sigma-Aldrich, F1804, dilution: 1:400), HA (Santa Cruz, sc-7392, dilution: 1:100), LC3 (MBL, PM036, dilution: 1:200), Hsp60 (Santa Cruz, sc-13115, dilution: 1:100), FIP200 (CST, 12436, dilution: 1:100), ATG16L1 (Proteintech, 19812, dilution: 1:100), WIPI2 (Proteintech, 15432, dilution: 1:100), ULK1 (Santa Cruz, Sc-390904, dilution: 1:50), MitoTracker Red FM (Thermo Fisher Scientific, M22425, dilution: 1:20000), MitoTracker Deep Red FM (Thermo Fisher Scientific, M22426, dilution: 1:20000), LysoTracker Deep Red FM (Thermo Fisher Scientific, L12492, dilution: 1:20,000), Goat anti-Rabbit (A10523) and anti-Mouse (A10524) IgG (H+L) Cross-Adsorbed Secondary Antibody, Cy5 (Thermo Fisher Scientific, dilution: 1:200), Cy3-AffiniPure Goat Anti-Rabbit (111-165-003) and Anti-Mouse (115-165-003) IgG (H+L) (Jackson, dilution: 1:200), FITC-AffiniPure Rabbit Anti-Goat IgG (H+L) FITC (Yeasen, 33707ES60, dilution: 1:200).

Techniques: Immunostaining, Transfection, Two Tailed Test

Journal: Nature Communications

Article Title: Phase separation of Nur77 mediates celastrol-induced mitophagy by promoting the liquidity of p62/SQSTM1 condensates

doi: 10.1038/s41467-021-26295-8

Figure Lengend Snippet: a GFP-Nur77 (2 μM) undergoes phase separation. The size of Nur77 droplets was analyzed. Data were presented as mean values ± SEM ( n = 3 independent experiments). Scale bar, 10 μm. b Top, changes in fluorescence intensity of GFP-Nur77 droplets after photobleaching were plotted over time. Bottom, representative images of fluorescence recovery. Data were presented as mean values ± SEM ( n = 3 independent experiments). Scale bar, 1.5 μm. c Fusion of GFP-Nur77 droplets in 10% PEG-3.35 K. Scale bar, 20 μm. d Live imaging of GFP-Nur77 in HeLa cells. Scale bar, 5 μm. e Time course analysis of GFP-Nur77 nuclear body recovery after photobleaching in HeLa cells. Representative images of fluorescence recovery are shown. Data were presented as mean values ± SEM ( n = 3 independent experiments). Scale bar, 1.5 μm. f Three-dimensional (3D) images of Nur77 nuclear assemblies. An enlarged view of inset is also shown. Scale bar, 5 μm. g Fixed imaging of Myc-Nur77 in HeLa cells. Scale bar, 5 μm. h Endogenous Nur77 displays nuclear puncta in HeLa cells revealed by immunostaining with anti-Nur77. Scale bar, 5 μm. Data represent at least three independent experiments. Source data are provided as a Source Data file.

Article Snippet: For immunostaining, the following antibodies were used: Nur77 for HeLa cell study (CST, 3960S, dilution: 1:100), Nur77 (M-210) (Santa Cruz, sc-5569, dilution: 1:50), Nur77 for mouse study (Affinity, DF7850, dilution:1:200), CD68 (Abcam, AB955, dilution: 1:200), SQSTM1/p62 (Abcam, AB56416, dilution: 1:400), Myc (9E10) (Santa Cruz, sc-40, dilution: 1:100), FLAG (Sigma-Aldrich, F1804, dilution: 1:400), HA (Santa Cruz, sc-7392, dilution: 1:100), LC3 (MBL, PM036, dilution: 1:200), Hsp60 (Santa Cruz, sc-13115, dilution: 1:100), FIP200 (CST, 12436, dilution: 1:100), ATG16L1 (Proteintech, 19812, dilution: 1:100), WIPI2 (Proteintech, 15432, dilution: 1:100), ULK1 (Santa Cruz, Sc-390904, dilution: 1:50), MitoTracker Red FM (Thermo Fisher Scientific, M22425, dilution: 1:20000), MitoTracker Deep Red FM (Thermo Fisher Scientific, M22426, dilution: 1:20000), LysoTracker Deep Red FM (Thermo Fisher Scientific, L12492, dilution: 1:20,000), Goat anti-Rabbit (A10523) and anti-Mouse (A10524) IgG (H+L) Cross-Adsorbed Secondary Antibody, Cy5 (Thermo Fisher Scientific, dilution: 1:200), Cy3-AffiniPure Goat Anti-Rabbit (111-165-003) and Anti-Mouse (115-165-003) IgG (H+L) (Jackson, dilution: 1:200), FITC-AffiniPure Rabbit Anti-Goat IgG (H+L) FITC (Yeasen, 33707ES60, dilution: 1:200).

Techniques: Fluorescence, Imaging, Immunostaining

Journal: Nature Communications

Article Title: Phase separation of Nur77 mediates celastrol-induced mitophagy by promoting the liquidity of p62/SQSTM1 condensates

doi: 10.1038/s41467-021-26295-8

Figure Lengend Snippet: a Intrinsic disorder tendency of Nur77. IDR intrinsically disordered region, DBD DNA-binding domain, LBD ligand-binding domain. b Schematic representation of Nur77 and its mutants. c In vitro phase separation of GFP-Nur77-IDR and GFP-Nur77-LBD (2 μM). Scale bar, 10 μm. d Quantification of Nur77 mutant droplets formation in absence of celastrol in HeLa cells. Data were presented as mean values ± SEM ( n = 3 independent experiments). e Representative images of Nur77 mutant droplets formation in absence of celastrol in HeLa cells. Scale bar, 10 μm. f Representative real-time images showing the formation and fusion of cytoplasmic GFP-Nur77 droplets after treatment with celastrol (2 μM) for 1 h in HeLa cells (see also Supplementary Movie 4). Right: quantification of the cytoplasmic retention of GFP-Nur77 protein. A two-tailed unpaired Student’s t -test was used for statistical analysis, and data were presented as mean values ± SEM ( n = 3 independent experiments). Scale bar, 10 μm. g Droplet formation of GFP-Nur77 and mutants in HeLa cells treated with the indicated compounds (2 μM). Left: Representative droplet images of transfected GFP-Nur77 and mutants. An enlarged view of the inset is also shown. Scale bar, 10 μm. Right: Quantification of droplet formation of GFP-Nur77 and mutants. Data represent at least three independent experiments. Source data are provided as Source Data file.

Article Snippet: For immunostaining, the following antibodies were used: Nur77 for HeLa cell study (CST, 3960S, dilution: 1:100), Nur77 (M-210) (Santa Cruz, sc-5569, dilution: 1:50), Nur77 for mouse study (Affinity, DF7850, dilution:1:200), CD68 (Abcam, AB955, dilution: 1:200), SQSTM1/p62 (Abcam, AB56416, dilution: 1:400), Myc (9E10) (Santa Cruz, sc-40, dilution: 1:100), FLAG (Sigma-Aldrich, F1804, dilution: 1:400), HA (Santa Cruz, sc-7392, dilution: 1:100), LC3 (MBL, PM036, dilution: 1:200), Hsp60 (Santa Cruz, sc-13115, dilution: 1:100), FIP200 (CST, 12436, dilution: 1:100), ATG16L1 (Proteintech, 19812, dilution: 1:100), WIPI2 (Proteintech, 15432, dilution: 1:100), ULK1 (Santa Cruz, Sc-390904, dilution: 1:50), MitoTracker Red FM (Thermo Fisher Scientific, M22425, dilution: 1:20000), MitoTracker Deep Red FM (Thermo Fisher Scientific, M22426, dilution: 1:20000), LysoTracker Deep Red FM (Thermo Fisher Scientific, L12492, dilution: 1:20,000), Goat anti-Rabbit (A10523) and anti-Mouse (A10524) IgG (H+L) Cross-Adsorbed Secondary Antibody, Cy5 (Thermo Fisher Scientific, dilution: 1:200), Cy3-AffiniPure Goat Anti-Rabbit (111-165-003) and Anti-Mouse (115-165-003) IgG (H+L) (Jackson, dilution: 1:200), FITC-AffiniPure Rabbit Anti-Goat IgG (H+L) FITC (Yeasen, 33707ES60, dilution: 1:200).

Techniques: Binding Assay, Ligand Binding Assay, In Vitro, Mutagenesis, Two Tailed Test, Transfection

Journal: Nature Communications

Article Title: Phase separation of Nur77 mediates celastrol-induced mitophagy by promoting the liquidity of p62/SQSTM1 condensates

doi: 10.1038/s41467-021-26295-8

Figure Lengend Snippet: a Immunofluorescence images of GFP-Nur77, mCherry-p62, and HA-Ub transfected in HeLa cells treated with or without celastrol. Data illustrate the colocalization of Nur77 with p62 and Ub in the presence of celastrol. Scale bar, 10 μm. b , c Interaction of indicated Nur77 or deubiquitinated mutant (K536R) and p62 was analyzed in HeLa cells treated with or without celastrol by co-immunoprecipitation (co-IP) assay. d , e Immunofluorescence images showing the effect of celastrol-induced Nur77 ubiquitination on mCherry-p62 droplet formation. Scale bar, 10 μm. f Representative images showing ubiquitination-dependent colocalization of Nur77 with p62 and mitochondria in HeLa cells treated with celastrol. Scale bar, 10 μm. Data represent at least three independent experiments. Source data are provided as Source Data file.

Article Snippet: For immunostaining, the following antibodies were used: Nur77 for HeLa cell study (CST, 3960S, dilution: 1:100), Nur77 (M-210) (Santa Cruz, sc-5569, dilution: 1:50), Nur77 for mouse study (Affinity, DF7850, dilution:1:200), CD68 (Abcam, AB955, dilution: 1:200), SQSTM1/p62 (Abcam, AB56416, dilution: 1:400), Myc (9E10) (Santa Cruz, sc-40, dilution: 1:100), FLAG (Sigma-Aldrich, F1804, dilution: 1:400), HA (Santa Cruz, sc-7392, dilution: 1:100), LC3 (MBL, PM036, dilution: 1:200), Hsp60 (Santa Cruz, sc-13115, dilution: 1:100), FIP200 (CST, 12436, dilution: 1:100), ATG16L1 (Proteintech, 19812, dilution: 1:100), WIPI2 (Proteintech, 15432, dilution: 1:100), ULK1 (Santa Cruz, Sc-390904, dilution: 1:50), MitoTracker Red FM (Thermo Fisher Scientific, M22425, dilution: 1:20000), MitoTracker Deep Red FM (Thermo Fisher Scientific, M22426, dilution: 1:20000), LysoTracker Deep Red FM (Thermo Fisher Scientific, L12492, dilution: 1:20,000), Goat anti-Rabbit (A10523) and anti-Mouse (A10524) IgG (H+L) Cross-Adsorbed Secondary Antibody, Cy5 (Thermo Fisher Scientific, dilution: 1:200), Cy3-AffiniPure Goat Anti-Rabbit (111-165-003) and Anti-Mouse (115-165-003) IgG (H+L) (Jackson, dilution: 1:200), FITC-AffiniPure Rabbit Anti-Goat IgG (H+L) FITC (Yeasen, 33707ES60, dilution: 1:200).

Techniques: Immunofluorescence, Transfection, Mutagenesis, Co-Immunoprecipitation Assay, Ubiquitin Proteomics

Journal: Nature Communications

Article Title: Phase separation of Nur77 mediates celastrol-induced mitophagy by promoting the liquidity of p62/SQSTM1 condensates

doi: 10.1038/s41467-021-26295-8

Figure Lengend Snippet: a –d Representative images showing colocalization of Nur77 or mutants with p62, mitochondria, and lysosome in HeLa cells after celastrol treatment. The blue arrow indicates line profiles of fluorescence intensities including Pearson’s correlation coefficients shown in b and d . A two-tailed unpaired Student’s t -test was used for statistical analysis, and data were presented as mean values ± SEM ( n = 20 biologically independent samples). Dotted box: higher magnification of indicated region. Scale bar, 10 μm. e Mutating K536 in Nur77 inhibits celastrol-induced interaction between p62 and LC3. HeLa cells transfected with the indicated expression plasmids were treated with or without 2 μM celastrol and 20 ng/mL TNFα. Interaction of Flag-p62 with GFP-LC3 was examined by co-IP assay. f Characterization of domain requirement of Nur77 for promoting celastrol-induced p62 interaction with LC3. HeLa cells transfected with the indicated Flag-p62, GFP-LC3, and GFP-Nur77 or mutant were treated with celastrol and TNFα for 1 h and analyzed for Flag-p62 interaction with GFP-LC3 by co-IP assay. Data represent at least three independent experiments. Source data are provided as Source Data file.

Article Snippet: For immunostaining, the following antibodies were used: Nur77 for HeLa cell study (CST, 3960S, dilution: 1:100), Nur77 (M-210) (Santa Cruz, sc-5569, dilution: 1:50), Nur77 for mouse study (Affinity, DF7850, dilution:1:200), CD68 (Abcam, AB955, dilution: 1:200), SQSTM1/p62 (Abcam, AB56416, dilution: 1:400), Myc (9E10) (Santa Cruz, sc-40, dilution: 1:100), FLAG (Sigma-Aldrich, F1804, dilution: 1:400), HA (Santa Cruz, sc-7392, dilution: 1:100), LC3 (MBL, PM036, dilution: 1:200), Hsp60 (Santa Cruz, sc-13115, dilution: 1:100), FIP200 (CST, 12436, dilution: 1:100), ATG16L1 (Proteintech, 19812, dilution: 1:100), WIPI2 (Proteintech, 15432, dilution: 1:100), ULK1 (Santa Cruz, Sc-390904, dilution: 1:50), MitoTracker Red FM (Thermo Fisher Scientific, M22425, dilution: 1:20000), MitoTracker Deep Red FM (Thermo Fisher Scientific, M22426, dilution: 1:20000), LysoTracker Deep Red FM (Thermo Fisher Scientific, L12492, dilution: 1:20,000), Goat anti-Rabbit (A10523) and anti-Mouse (A10524) IgG (H+L) Cross-Adsorbed Secondary Antibody, Cy5 (Thermo Fisher Scientific, dilution: 1:200), Cy3-AffiniPure Goat Anti-Rabbit (111-165-003) and Anti-Mouse (115-165-003) IgG (H+L) (Jackson, dilution: 1:200), FITC-AffiniPure Rabbit Anti-Goat IgG (H+L) FITC (Yeasen, 33707ES60, dilution: 1:200).

Techniques: Fluorescence, Two Tailed Test, Transfection, Expressing, Co-Immunoprecipitation Assay, Mutagenesis

Journal: Nature Communications

Article Title: Phase separation of Nur77 mediates celastrol-induced mitophagy by promoting the liquidity of p62/SQSTM1 condensates

doi: 10.1038/s41467-021-26295-8

Figure Lengend Snippet: a Schematic representation of p62 and mutants and their interaction with Nur77. PB1 Phox/Bem1p protein-protein binding domain. ZZ zinc-finger domain, TB TRAF6 binding domain, UBA ubiquitin-associated domain. b – d Interaction of Nur77 and p62, as well as their mutants, was analyzed in HeLa cells treated with or without celastrol by co-IP assay. e Multivalent interaction between Nur77 and p62. The interaction between the IDR of Nur77 and PB1 of P62 is ligand-independent (red), whereas the interaction between LBD of Nur77 and UBA of p62 depends on celastrol that triggers Nur77-LBD ubiquitination (pink). f Immunofluorescence images showing colocalization of GFP-Nur77-IDR with mCherry-p62 or mCherry-p62-PB1 after treatment with or without celastrol. Scale bar, 10 μm. g FRAP analysis of the effect of Nur77-IDR in regulating p62 mobility in HeLa cells. Data were presented as mean values ± SEM ( n = 3 independent experiments). Scale bar, 1.5 μm. h Representative images showing the effect of GFP-Nur77-IDR on the filamentous structures of mCherry-p62-PB1 when mCherry-p62-PB1 was incubated with GFP or GFP-Nur77-IDR at intermediate molar ratio (1:2). Scale bar, 10 μm. i FRAP analysis of the effect of GFP-Nur77-IDR on mCherry-p62-PB1 mobility in vitro. Two-tailed unpaired Student’s t -test was used for statistical analysis, and data were presented as mean values ± SEM ( n = 3 independent experiments). Data represent at least three independent experiments. Source data are provided as Source Data file.

Article Snippet: For immunostaining, the following antibodies were used: Nur77 for HeLa cell study (CST, 3960S, dilution: 1:100), Nur77 (M-210) (Santa Cruz, sc-5569, dilution: 1:50), Nur77 for mouse study (Affinity, DF7850, dilution:1:200), CD68 (Abcam, AB955, dilution: 1:200), SQSTM1/p62 (Abcam, AB56416, dilution: 1:400), Myc (9E10) (Santa Cruz, sc-40, dilution: 1:100), FLAG (Sigma-Aldrich, F1804, dilution: 1:400), HA (Santa Cruz, sc-7392, dilution: 1:100), LC3 (MBL, PM036, dilution: 1:200), Hsp60 (Santa Cruz, sc-13115, dilution: 1:100), FIP200 (CST, 12436, dilution: 1:100), ATG16L1 (Proteintech, 19812, dilution: 1:100), WIPI2 (Proteintech, 15432, dilution: 1:100), ULK1 (Santa Cruz, Sc-390904, dilution: 1:50), MitoTracker Red FM (Thermo Fisher Scientific, M22425, dilution: 1:20000), MitoTracker Deep Red FM (Thermo Fisher Scientific, M22426, dilution: 1:20000), LysoTracker Deep Red FM (Thermo Fisher Scientific, L12492, dilution: 1:20,000), Goat anti-Rabbit (A10523) and anti-Mouse (A10524) IgG (H+L) Cross-Adsorbed Secondary Antibody, Cy5 (Thermo Fisher Scientific, dilution: 1:200), Cy3-AffiniPure Goat Anti-Rabbit (111-165-003) and Anti-Mouse (115-165-003) IgG (H+L) (Jackson, dilution: 1:200), FITC-AffiniPure Rabbit Anti-Goat IgG (H+L) FITC (Yeasen, 33707ES60, dilution: 1:200).

Techniques: Protein Binding, Binding Assay, Ubiquitin Proteomics, Co-Immunoprecipitation Assay, Immunofluorescence, Incubation, In Vitro, Two Tailed Test

Journal: Nature Communications

Article Title: Phase separation of Nur77 mediates celastrol-induced mitophagy by promoting the liquidity of p62/SQSTM1 condensates

doi: 10.1038/s41467-021-26295-8

Figure Lengend Snippet: The phase separation of Nur77 and p62/SQSTM1 triggered by their multivalent interaction sequesters damaged mitochondria and directs cargo mitochondria to the autophagic machinery. DBD DNA-binding domain, LBD ligand-binding domain, IDR intrinsically disordered region, PB1 Phor and Bem1p, UBA ubiquitin-associating, Ub ubiquitin.

Article Snippet: For immunostaining, the following antibodies were used: Nur77 for HeLa cell study (CST, 3960S, dilution: 1:100), Nur77 (M-210) (Santa Cruz, sc-5569, dilution: 1:50), Nur77 for mouse study (Affinity, DF7850, dilution:1:200), CD68 (Abcam, AB955, dilution: 1:200), SQSTM1/p62 (Abcam, AB56416, dilution: 1:400), Myc (9E10) (Santa Cruz, sc-40, dilution: 1:100), FLAG (Sigma-Aldrich, F1804, dilution: 1:400), HA (Santa Cruz, sc-7392, dilution: 1:100), LC3 (MBL, PM036, dilution: 1:200), Hsp60 (Santa Cruz, sc-13115, dilution: 1:100), FIP200 (CST, 12436, dilution: 1:100), ATG16L1 (Proteintech, 19812, dilution: 1:100), WIPI2 (Proteintech, 15432, dilution: 1:100), ULK1 (Santa Cruz, Sc-390904, dilution: 1:50), MitoTracker Red FM (Thermo Fisher Scientific, M22425, dilution: 1:20000), MitoTracker Deep Red FM (Thermo Fisher Scientific, M22426, dilution: 1:20000), LysoTracker Deep Red FM (Thermo Fisher Scientific, L12492, dilution: 1:20,000), Goat anti-Rabbit (A10523) and anti-Mouse (A10524) IgG (H+L) Cross-Adsorbed Secondary Antibody, Cy5 (Thermo Fisher Scientific, dilution: 1:200), Cy3-AffiniPure Goat Anti-Rabbit (111-165-003) and Anti-Mouse (115-165-003) IgG (H+L) (Jackson, dilution: 1:200), FITC-AffiniPure Rabbit Anti-Goat IgG (H+L) FITC (Yeasen, 33707ES60, dilution: 1:200).

Techniques: Binding Assay, Ligand Binding Assay, Ubiquitin Proteomics

Journal: Biochemical and biophysical research communications

Article Title: Tyrosine kinase activity of EphA2 promotes its S897 phosphorylation and glioblastoma cell proliferation.

doi: 10.1016/j.bbrc.2018.04.020

Figure Lengend Snippet: Fig. 1. EphA2 S897 phosphorylation is enhanced by its tyrosine kinase activity. A, D. The EphA2 constructs used in this study. LBD, ligand binding domain; C, Cysteine rich domain; FN, fibronectinⅢrepeats; TM, transmembrane; KD, kinase domain; SAM, sterile- a-motif. Numbers indicate amino acid position within the sequence. B, F. Cell lysates from HEK293T cells transfected with the indicated plasmids were immunoblotted with the indicated antibodies. C. Densitometry analysis was performed with ImageJ software, and the pS897 EphA2/EphA2 ratio was determined. Data are the means ± SD of three independent experiments (*p < 0.05; t-test). E. HeLa cells transfected with Control or Flag-tagged EphA2-ICD were treated with EGF (100 ng/ml) for 10 min, and the cell lysates were analyzed by immu- noblotting with the indicated antibodies.

Article Snippet: We used the following antibodies in this study: rabbit monoclonal antibodies against EphA2 (D4A2), S897 phosphoEphA2 (D9A1), Y588 phospho-EphA2 (D7X2L), ERK, and T202/ Y204 phospho-ERK, (Cell Signaling Technology); a mouse monoclonal antibody against Flag (M2) (Sigma); a mouse monoclonal antibody against BrdU (3D4) (BD Biosciences); secondary antibodies conjugated to horseradish peroxidase (DAKO); Alexa Fluor 488-conjugated anti-GFP and Alexa Fluor 594-conjugated goat anti-mouse IgG (Thermo Fisher Scientific).

Techniques: Phospho-proteomics, Activity Assay, Construct, Ligand Binding Assay, Sterility, Sequencing, Transfection, Software, Control

Journal: Biochemical and biophysical research communications

Article Title: Tyrosine kinase activity of EphA2 promotes its S897 phosphorylation and glioblastoma cell proliferation.

doi: 10.1016/j.bbrc.2018.04.020

Figure Lengend Snippet: Fig. 2. EphA2 induces ERK activation through its tyrosine kinase activity. A, C. Cell lysates from HEK293T cells transfected with the indicated plasmids were immunoblotted with the indicated antibodies. B, D. Densitometry analysis was performed with ImageJ software and the pERK/ERK ratio was determined. Data are the means ± SD of three independent experiments (**p < 0.01, ***p < 0.001, one-way ANOVA, Tukey's HSD post hoc test). E. U-251 cells were treated with control-Fc or ephrinA1-Fc (1 mg/ml) for 24 h, and the cell lysates were analyzed by immunoblotting with the indicated antibodies. F. Densitometry analysis was performed with ImageJ software and the pERK/ERK ratio was determined. Data are the means ± SD of three independent experiments (**p < 0.01, ***p < 0.001, one-way ANOVA, Tukey's HSD post hoc test).

Article Snippet: We used the following antibodies in this study: rabbit monoclonal antibodies against EphA2 (D4A2), S897 phosphoEphA2 (D9A1), Y588 phospho-EphA2 (D7X2L), ERK, and T202/ Y204 phospho-ERK, (Cell Signaling Technology); a mouse monoclonal antibody against Flag (M2) (Sigma); a mouse monoclonal antibody against BrdU (3D4) (BD Biosciences); secondary antibodies conjugated to horseradish peroxidase (DAKO); Alexa Fluor 488-conjugated anti-GFP and Alexa Fluor 594-conjugated goat anti-mouse IgG (Thermo Fisher Scientific).

Techniques: Activation Assay, Activity Assay, Transfection, Software, Control, Western Blot

Journal: Biochemical and biophysical research communications

Article Title: Tyrosine kinase activity of EphA2 promotes its S897 phosphorylation and glioblastoma cell proliferation.

doi: 10.1016/j.bbrc.2018.04.020

Figure Lengend Snippet: Fig. 3. ERK activation is required for EphA2-induced S897 phosphorylation. A. HEK293T cells were transfected with control or EphA2-WT, and treated with U0126 (20 mM) for 15 h. Cell lysates were analyzed by immunoblotting with the indicated antibodies. B. HEK293T cells were transfected with control or EphA2-WT, and treated with MK2206 (1 mM) or LY294002 (20 mM) for 15 h. Cell lysates were analyzed by immunoblotting with the indicated antibodies.

Article Snippet: We used the following antibodies in this study: rabbit monoclonal antibodies against EphA2 (D4A2), S897 phosphoEphA2 (D9A1), Y588 phospho-EphA2 (D7X2L), ERK, and T202/ Y204 phospho-ERK, (Cell Signaling Technology); a mouse monoclonal antibody against Flag (M2) (Sigma); a mouse monoclonal antibody against BrdU (3D4) (BD Biosciences); secondary antibodies conjugated to horseradish peroxidase (DAKO); Alexa Fluor 488-conjugated anti-GFP and Alexa Fluor 594-conjugated goat anti-mouse IgG (Thermo Fisher Scientific).

Techniques: Activation Assay, Phospho-proteomics, Transfection, Control, Western Blot

Journal: Biochemical and biophysical research communications

Article Title: Tyrosine kinase activity of EphA2 promotes its S897 phosphorylation and glioblastoma cell proliferation.

doi: 10.1016/j.bbrc.2018.04.020

Figure Lengend Snippet: Fig. 4. EphA2 promotes glioblastoma cell proliferation through its tyrosine kinase activity. A, B. A172 cells were transfected with YFP and the indicated plasmids. Then cells were stained with anti-BrdU and anti-GFP antibodies. The number of BrdU-positive and/or YFP- positive cells was counted, and the percentage of BrdU-labeled YFP-positive cells in the total number of YFP-positive cells (BrdU þ YFPþ/YFPþ) was shown. Data are the means ± SD of seven independent experiments (**p < 0.01, ***p < 0.001, one-way ANOVA, Tukey's HSD post hoc test). C. Overexpression of EphA2 stimulates its tyrosine kinase activity and induces ERK activation, which results in S897 phosphorylation, leading to the promotion of glioblastoma cell proliferation.

Article Snippet: We used the following antibodies in this study: rabbit monoclonal antibodies against EphA2 (D4A2), S897 phosphoEphA2 (D9A1), Y588 phospho-EphA2 (D7X2L), ERK, and T202/ Y204 phospho-ERK, (Cell Signaling Technology); a mouse monoclonal antibody against Flag (M2) (Sigma); a mouse monoclonal antibody against BrdU (3D4) (BD Biosciences); secondary antibodies conjugated to horseradish peroxidase (DAKO); Alexa Fluor 488-conjugated anti-GFP and Alexa Fluor 594-conjugated goat anti-mouse IgG (Thermo Fisher Scientific).

Techniques: Activity Assay, Transfection, Staining, Labeling, Over Expression, Activation Assay, Phospho-proteomics

Journal: The Journal of Biological Chemistry

Article Title: A cancer mutation promotes EphA4 oligomerization and signaling by altering the conformation of the SAM domain

doi: 10.1016/j.jbc.2021.100876

Figure Lengend Snippet: Correlation of EphA4 expression with patient survival and EphA4 mutations in melanoma. A , the correlation between high (top 15%) EphA4 mRNA expression and decreased overall patient survival does not reach statistical significance when considering all melanoma tumors. B , high EphA4 expression in the subset of tumors with highest (top 15%) mRNA expression of one or more of the five ephrinA ligands significantly correlates with decreased patient survival. In both A and B , the 15% of tumor samples with highest EphA4 expression were compared to the 85% remaining tumor samples. B includes for each ephrinA ligand the 15% of tumors with highest expression, for a total of ∼45% of all the tumors. mRNA expression z-scores relative to all samples (log RNA Seq V2 RSEM) from the TCGA Firehose Legacy skin cutaneous melanoma dataset (n = 472 tumor samples with mRNA expression data) were used for analysis. Median survival times are indicated in the graphs and p values were calculated using the log-rank Mantel–Cox test. C , the location of the eight EphA4 melanoma mutations analyzed is shown in relation to the EphA4 domain structure. The mutations were selected for further investigation from 12 skin melanoma studies available in the cBioPortal website ( cbioportal.org ). The height of the black vertical lines indicates the number of tumors with that particular mutation. The colored dots above the name of each mutation indicate the prediction of functional significance according to three prediction programs: Mutation Assessor, SIFT and PolyPhen-2 ( cbioportal.org ). The EphA4 signal peptide and transmembrane helix are shown in light gray and linkers are shown in dark gray , including the juxtamembrane segment containing the P605S mutation. D , EphA4 WT, the eight EphA4 mutants, and EGFP as a control were transiently expressed in HEK293 cells and cell lysates were probed by immunoblotting with antibodies to phosphotyrosine (pTyr) and to EphA4. The bar graph shows averages and standard errors from quantifications of three experiments (individual values from each experiment are shown as dots ). ∗∗∗ p < 0.001 and ∗∗∗∗ p < 0.0001 for the comparison with WT by one-way ANOVA. EGF, epidermal growth factor-like domain; FNIII, fibronectin type III domain; LBD, ligand-binding domain; kinase, kinase domain; SAM, sterile alpha motif domain; sushi, sushi domain.

Article Snippet: After semidry transfer, the immobilon membranes were blocked with 5% bovine serum albumin in 0.1% Tween-20 in TBS (150 mM NaCl, 50 mM TrisHCl pH 7.5) for 1 h and then incubated overnight at 4 °C in blocking buffer containing primary antibodies recognizing EphA4 (BD Biosciences, 610471; 1:1000; D , , A – C , B and A , an affinity-purified rabbit polyclonal EphA4 antibody generated using a peptide corresponding to the 11 C-terminal amino acids of human and mouse EphA4 ( ) and used at 1 μg/ml in D and A ); EphA3 pY779 (Cell Signaling Technology, 8862; 1:1000 dilution), which also recognizes EphA4 pY779; β-tubulin (Cell Signaling Technology, 2128; 1:25,000 dilution); the FLAG tag (Sigma, F1804; 1:1000 dilution) and an HRP-conjugated antibody recognizing phosphotyrosine (Cell Signaling Technology, 5465; 1:2000).

Techniques: Expressing, RNA Sequencing, Mutagenesis, Functional Assay, Control, Western Blot, Comparison, Ligand Binding Assay, Sterility

Journal: The Journal of Biological Chemistry

Article Title: A cancer mutation promotes EphA4 oligomerization and signaling by altering the conformation of the SAM domain

doi: 10.1016/j.jbc.2021.100876

Figure Lengend Snippet: The L920F mutation in the EphA4 SAM domain promotes receptor tyrosine phosphorylation and activation. A , HEK293 cells were transiently transfected with constructs encoding EphA4 WT, the EphA4 L920F mutant, or EGFP as a control. Cell lysates were probed by immunoblotting with antibodies to phosphotyrosine (pTyr), the Y602 and Y779 EphA4 phosphorylation sites, EphA4, and vimentin as a loading control. B , HEK293 cells were transiently transfected as indicated and treated for 10 min with 0.5 μg/ml ephrinA5-Fc (+) or Fc as a control (−). Cell lysates were probed by immunoblotting with antibodies to the Y779 EphA4 phosphorylation site (short and long exposures are shown), EphA4, and β-tubulin as a loading control. C , HEK293 cells were stably transfected with constructs encoding FLAG-tagged EphA4 WT or L920F mutant, or EGFP as a control, and treated for 10 min with 0.5 μg/ml ephrinA5-Fc (+) or Fc as a control (−). FLAG immmunoprecipitates and cell lysates were probed by immunoblotting with the indicated antibodies. D , HEK293 cells were transiently transfected with constructs encoding Strep-tagged EphA4 WT or L920F mutant with or without FLAG-tagged NCK2. FLAG immunoprecipitates and cell lysates were probed by immunoblotting with the indicated antibodies.

Article Snippet: After semidry transfer, the immobilon membranes were blocked with 5% bovine serum albumin in 0.1% Tween-20 in TBS (150 mM NaCl, 50 mM TrisHCl pH 7.5) for 1 h and then incubated overnight at 4 °C in blocking buffer containing primary antibodies recognizing EphA4 (BD Biosciences, 610471; 1:1000; D , , A – C , B and A , an affinity-purified rabbit polyclonal EphA4 antibody generated using a peptide corresponding to the 11 C-terminal amino acids of human and mouse EphA4 ( ) and used at 1 μg/ml in D and A ); EphA3 pY779 (Cell Signaling Technology, 8862; 1:1000 dilution), which also recognizes EphA4 pY779; β-tubulin (Cell Signaling Technology, 2128; 1:25,000 dilution); the FLAG tag (Sigma, F1804; 1:1000 dilution) and an HRP-conjugated antibody recognizing phosphotyrosine (Cell Signaling Technology, 5465; 1:2000).

Techniques: Mutagenesis, Phospho-proteomics, Activation Assay, Transfection, Construct, Control, Western Blot, Stable Transfection

Journal: The Journal of Biological Chemistry

Article Title: A cancer mutation promotes EphA4 oligomerization and signaling by altering the conformation of the SAM domain

doi: 10.1016/j.jbc.2021.100876

Figure Lengend Snippet: All-atom MD simulations suggest that the EphA4 L920F mutation introduces local and global structural perturbations. A , structure of the EphA4 WT SAM domain. ( left ; PDB ID: 1B0X ; ) and model of the L920F mutant ( right ), obtained by direct substitution of L920 with phenylalanine, in ribbon representation with the indicated residues shown as sticks and as a molecular surface. The model illustrates how W919 and F932 clash with a phenylalanine at position 920. B , representative structures obtained from MD simulations of the EphA4 WT SAM domain ( left ) and EphA4 L920F SAM domain ( right ) are aligned with the EphA4 SAM domain crystal structure ( gray ) by minimizing backbone RMSD values. C , the RMSF values calculated using the crystal structure coordinates as the reference are plotted for each residue in the EphA4 WT and L920F SAM domains over the course of the MD simulations, each averaged over three replicates. A schematic of the SAM domain helix positions is shown above the figure. D , dynamic network analysis for the EphA4 WT and L920F mutant SAM domain structures. Nodes, indicated by spheres , highlight the α-carbon atoms and the thickness of the edges (lines connecting the nodes) is proportional to the correlation of atomic motion in space and time. The WT L920 or mutant F920 is rendered with atoms as spheres. Six communities (sets of residues that exhibit coordinated motion) are shown in different colors: community 1 in yellow , community 2 in olive , community 3 in purple , community 4 in orange , community 5 in green , and community 6 in gray . Edges drawn within the same community are colored according to that community and edges drawn between nodes of different communities are colored black . The α-helices (H1 through H5) are indicated and the portion of H1 circled in red in the WT SAM domain is also shown as an enlargement to highlight the different communities in this α-helix.

Article Snippet: After semidry transfer, the immobilon membranes were blocked with 5% bovine serum albumin in 0.1% Tween-20 in TBS (150 mM NaCl, 50 mM TrisHCl pH 7.5) for 1 h and then incubated overnight at 4 °C in blocking buffer containing primary antibodies recognizing EphA4 (BD Biosciences, 610471; 1:1000; D , , A – C , B and A , an affinity-purified rabbit polyclonal EphA4 antibody generated using a peptide corresponding to the 11 C-terminal amino acids of human and mouse EphA4 ( ) and used at 1 μg/ml in D and A ); EphA3 pY779 (Cell Signaling Technology, 8862; 1:1000 dilution), which also recognizes EphA4 pY779; β-tubulin (Cell Signaling Technology, 2128; 1:25,000 dilution); the FLAG tag (Sigma, F1804; 1:1000 dilution) and an HRP-conjugated antibody recognizing phosphotyrosine (Cell Signaling Technology, 5465; 1:2000).

Techniques: Mutagenesis, Residue

Journal: The Journal of Biological Chemistry

Article Title: A cancer mutation promotes EphA4 oligomerization and signaling by altering the conformation of the SAM domain

doi: 10.1016/j.jbc.2021.100876

Figure Lengend Snippet: The L920F mutation induces EphA4 oligomerization. A , HEK293 cells were transiently cotransfected with FLAG- or Strep-tagged EphA4 WT or L920F mutant. Cell lysates were subjected to pull-down with Strep-Tactin beads and the proteins bound to the beads were eluted, split into two aliquots and each aliquot was probed by immunoblotting with antibodies to the FLAG tag to detect FLAG-EphA4 or to the Strep tag to detect Strep-EphA4. Different aliquots of the lysates were also probed as indicated. B , HEK293 cells were transiently cotransfected with EphA4 (WT or L920F mutant) fused to C-terminal mTURQ or EYFP fluorescent proteins. Cell lysates were probed by immunoblotting with antibodies to phosphotyrosine (pTyr), the Y779 EphA4 phosphorylation site, and EphA4. C , FRET efficiencies measured for EphA4-mTURQ and EphA4-EYFP, WT, and L920F mutant, in the absence of ligand using the FSI-FRET method. D , mean square error (MSE) values for best-fit oligomerization models ranging from monomers ( n = 1) to hexamers ( n = 6). The minimum MSE indicates the model that best describes the data. E , FIF measurements performed in HEK293 cells expressing EphA4 WT or L920F. Shown are histograms of the measured molecular brightness (ε), which scales with the oligomer size. The brightness values corresponding to the histogram maxima are indicated by the dotted lines . F , dimeric or oligomeric fractions calculated from the FSI-FRET data are plotted as a function of total receptor concentration for EphA4 WT and L920F. The symbols represent the binned oligomeric fractions and their standard errors. The solid lines represent the best fit curves for monomer–dimer or monomer–oligomer equilibrium. Although lower maximal EphA4 L920F acceptor expression was achieved than for WT (panel C ), a complete oligomerization curve was obtained. The dissociation constant (or apparent dissociation constant in the oligomer case) is determined as the receptor concentration at which the oligomeric fraction is 0.5 (50%; see Table 1 ). G , high-concentration FIF histograms for EphA4 WT and L920F mutant, generated by using the data from E only for receptor concentrations higher than 3000 receptors/μm 2 . By removing low-concentration data, the monomer populations are not significantly present and thus the maximum of the histogram more accurately indicates receptor oligomer size, ε = 2 (dimer) for EphA4 WT and ε = 3 (trimer) for the L920F mutant. H , FRET efficiencies as a function of acceptor concentration measured for EphA4 WT and L920F in the presence of the ephrinA5-Fc ligand. I , MSE values for EphA4 WT and EphA4 L920F with ephrinA5-Fc. J , FIF histograms for EphA4 WT and L920F mutant in the presence of ephrinA5-Fc. The maxima of the histograms are indicated by dotted line s . K , oligomerization curves for EphA4 WT and the EphA4 L920F mutant in the presence of ephrinA5-Fc (see Table 1 ). Although lower maximal EphA4 L920F acceptor expression was achieved than for WT (panel H ), a complete oligomerization curve was obtained. FRET, Föster resonance energy transfer.

Article Snippet: After semidry transfer, the immobilon membranes were blocked with 5% bovine serum albumin in 0.1% Tween-20 in TBS (150 mM NaCl, 50 mM TrisHCl pH 7.5) for 1 h and then incubated overnight at 4 °C in blocking buffer containing primary antibodies recognizing EphA4 (BD Biosciences, 610471; 1:1000; D , , A – C , B and A , an affinity-purified rabbit polyclonal EphA4 antibody generated using a peptide corresponding to the 11 C-terminal amino acids of human and mouse EphA4 ( ) and used at 1 μg/ml in D and A ); EphA3 pY779 (Cell Signaling Technology, 8862; 1:1000 dilution), which also recognizes EphA4 pY779; β-tubulin (Cell Signaling Technology, 2128; 1:25,000 dilution); the FLAG tag (Sigma, F1804; 1:1000 dilution) and an HRP-conjugated antibody recognizing phosphotyrosine (Cell Signaling Technology, 5465; 1:2000).

Techniques: Mutagenesis, Western Blot, FLAG-tag, Strep-tag, Phospho-proteomics, Expressing, Concentration Assay, Generated, Förster Resonance Energy Transfer

Journal: The Journal of Biological Chemistry

Article Title: A cancer mutation promotes EphA4 oligomerization and signaling by altering the conformation of the SAM domain

doi: 10.1016/j.jbc.2021.100876

Figure Lengend Snippet: Summary of FSI-FRET experiments

Article Snippet: After semidry transfer, the immobilon membranes were blocked with 5% bovine serum albumin in 0.1% Tween-20 in TBS (150 mM NaCl, 50 mM TrisHCl pH 7.5) for 1 h and then incubated overnight at 4 °C in blocking buffer containing primary antibodies recognizing EphA4 (BD Biosciences, 610471; 1:1000; D , , A – C , B and A , an affinity-purified rabbit polyclonal EphA4 antibody generated using a peptide corresponding to the 11 C-terminal amino acids of human and mouse EphA4 ( ) and used at 1 μg/ml in D and A ); EphA3 pY779 (Cell Signaling Technology, 8862; 1:1000 dilution), which also recognizes EphA4 pY779; β-tubulin (Cell Signaling Technology, 2128; 1:25,000 dilution); the FLAG tag (Sigma, F1804; 1:1000 dilution) and an HRP-conjugated antibody recognizing phosphotyrosine (Cell Signaling Technology, 5465; 1:2000).

Techniques:

Journal: The Journal of Biological Chemistry

Article Title: A cancer mutation promotes EphA4 oligomerization and signaling by altering the conformation of the SAM domain

doi: 10.1016/j.jbc.2021.100876

Figure Lengend Snippet: In silico docking predicts two stable interfaces for the EphA4 L920F SAM domain. A , ten EphA4 L920F SAM dimer structures generated by ClusPro were optimized by creating 2000 decoys for each with PyRosetta. Interface scores for the decoys are plotted as a function of the RMSD value calculated relative to the initial ClusPro structure “0” (ClusPro outputs are numbered 0–9). Each of the ten sets of decoys is shown in a different color . The lowest-energy decoys from each set represent the optimized dimer structures. Two lowest-energy dimer structures for the EphA4 L920F SAM domain, referred to as AB ( orange ) and CD ( yellow ), are indicated by arrows . The decoy with the lowest interface score for the set of structures colored in yellow was considered an outlier (see Fig. S5 ), so the second lowest was selected. B , model of an EphA4 L920F SAM domain trimer that engages both AB and CD interfaces. The three SAM domains in the trimer are shown in orange (molecule A), yellow (molecule D), and gray (molecule B/C). Residue F920 is shown in red as sticks and as a molecular surface. Residue H945, shown in green as sticks and as a molecular surface, stabilizes both interfaces (see Table S1 ) by engaging H961 ( purple sticks and molecular surface) in molecule B/C and S967/S968 ( purple sticks and molecular surface) in molecule D. C , ten sets of EphA4 WT decoys were generated as described in A . A single lowest-energy structure, indicated by an asterisk , was identified for the EphA4 WT SAM domain ( green ). However, this model did not match some experimental data and therefore the second lowest structure ( cyan ), indicated by an arrow , was selected for further analysis. D , structural representation of the second lowest-energy EphA4 WT dimer structure in which the two SAM domains (A and B) are indicated in cyan and gray . Residue L920 is indicated in blue and residue H945 in yellow , both shown as sticks and as a molecular surface. This model is structurally similar to the crystallographic dimer shown in Fig. S6 C .

Article Snippet: After semidry transfer, the immobilon membranes were blocked with 5% bovine serum albumin in 0.1% Tween-20 in TBS (150 mM NaCl, 50 mM TrisHCl pH 7.5) for 1 h and then incubated overnight at 4 °C in blocking buffer containing primary antibodies recognizing EphA4 (BD Biosciences, 610471; 1:1000; D , , A – C , B and A , an affinity-purified rabbit polyclonal EphA4 antibody generated using a peptide corresponding to the 11 C-terminal amino acids of human and mouse EphA4 ( ) and used at 1 μg/ml in D and A ); EphA3 pY779 (Cell Signaling Technology, 8862; 1:1000 dilution), which also recognizes EphA4 pY779; β-tubulin (Cell Signaling Technology, 2128; 1:25,000 dilution); the FLAG tag (Sigma, F1804; 1:1000 dilution) and an HRP-conjugated antibody recognizing phosphotyrosine (Cell Signaling Technology, 5465; 1:2000).

Techniques: In Silico, Generated, Residue

Journal: The Journal of Biological Chemistry

Article Title: A cancer mutation promotes EphA4 oligomerization and signaling by altering the conformation of the SAM domain

doi: 10.1016/j.jbc.2021.100876

Figure Lengend Snippet: The H945E mutation destabilizes EphA4 L920F trimers. A , HEK293 cells were transiently transfected with constructs encoding EphA4 WT, the L920F single mutant, the L920F-H945E double mutant or EGFP as a control. Cell lysates were probed by immunoblotting with antibodies recognizing phosphotyrosine (pTyr), the Y779 phosphorylated motif (pY779), and EphA4. The bar graph shows averages and standard errors from quantifications of three experiments (individual values from each experiment are shown as dots ). ∗ p < 0.05 for the comparison with WT = 1 by one-sample t test. B , FRET efficiencies measured for the EphA4 L920F-H945E double mutant in HEK293T cells and compared with those for the L920F mutant. C , MSE values calculated for EphA4 L920F-H945E. D , oligomeric fractions calculated from the FSI-FRET data are plotted as a function of total receptor concentration for the EphA4 L920F-H945E double mutant compared with the L920F mutant (see Table 1 ). E , FRET efficiencies measured for EphA4 L920F-H945E in the presence of ephrinA5-Fc as a function of acceptor concentration and compared with the EphA4 L920F mutant. F , MSE values for EphA4 L920F-H945E in the presence of ligand. G , the oligomerization curve calculated from the FSI-FRET data for EphA4 L920F-H945E in the presence of ephrinA5-Fc compared with that for EphA4 L920F (see Table 1 ). H , FRET efficiencies measured for the EphA4 H945E mutant as a function of acceptor concentration and compared with EphA4 WT. I , MSE values calculated for EphA4 H945E. J , the oligomerization curve calculated from the FSI-FRET data for the EphA4 H945E mutant and compared with EphA4 WT (see Table 1 ). FRET, Föster resonance energy transfer.

Article Snippet: After semidry transfer, the immobilon membranes were blocked with 5% bovine serum albumin in 0.1% Tween-20 in TBS (150 mM NaCl, 50 mM TrisHCl pH 7.5) for 1 h and then incubated overnight at 4 °C in blocking buffer containing primary antibodies recognizing EphA4 (BD Biosciences, 610471; 1:1000; D , , A – C , B and A , an affinity-purified rabbit polyclonal EphA4 antibody generated using a peptide corresponding to the 11 C-terminal amino acids of human and mouse EphA4 ( ) and used at 1 μg/ml in D and A ); EphA3 pY779 (Cell Signaling Technology, 8862; 1:1000 dilution), which also recognizes EphA4 pY779; β-tubulin (Cell Signaling Technology, 2128; 1:25,000 dilution); the FLAG tag (Sigma, F1804; 1:1000 dilution) and an HRP-conjugated antibody recognizing phosphotyrosine (Cell Signaling Technology, 5465; 1:2000).

Techniques: Mutagenesis, Transfection, Construct, Control, Western Blot, Comparison, Concentration Assay, Förster Resonance Energy Transfer

Journal: Nature microbiology

Article Title: Spatiotemporal proteomics uncovers cathepsin-dependent macrophage cell death during Salmonella infection

doi: 10.1038/s41564-020-0736-7

Figure Lengend Snippet: Nuclear cathepsin translocation occurs already from 8 hpi, is independent of cathepsin activity and does not result in increased Histone 3 cleavage. a) Time course of nuclear cathepsin activity. Nuclei and S Tm were enriched by sequential 50 x g and 8,000 x g centrifugation steps, respectively. RAW264.7 cells were infected with wildtype S Tm 14028s and treated with DCG04-Bodipy-FLike (5μM) for 4 hours prior to harvesting. Samples were separated by SDS-PAGE and visualised using a fluorescent scanner (Ex 405 nm/Em 520 nm), followed by immunoblotting for the soluble cytoplasmic protein GAPDH, the bacterial protein RpoD and Coomassie staining for histones as a loading control for nuclear extracts. L = lysatome, N = nucleome, S Tm = S Tm enriched fraction. Experiment was performed once. b) RAW264.7 cells were infected with wildtype S Tm at MOI 100:1 and harvested at 20 hpi. Whole cell lysates were immunoblotted with the CtsL specific cleavage product of H3 (H3.cs1) 16 and histone 3 (H3) as loading control. The experiment was performed in biological duplicate per condition and were analysed in adjacent lanes. c) Nuclear extracts from RAW264.7 cells either mock infected, infected with wildtype S Tm or heat killed wildtype ( S Tm-HK) for 20 hours. CA-074-Me was present throughout the infection experiment at the indicated concentrations. Nuclear extracts were analysed by immunoblot for CtsB (1:2,000), Lamin A (1:5,000) and histones by Coommassie stain (see for second replicate data).

Article Snippet: In brief, 10 U/100 μL of recombinant Caspase-11 (mouse, Enzo life sciences; BML-SE155-5000) in caspase activity buffer (200 mM NaCl, 50 mM HEPES pH 8.0, 50 mM KCl, 10 mM DTT) supplemented with 100 μMAcLEHD-afc (Santa Cruz; sc-311277) was incubated in the presence of CA-074-Me, E64d (Sigma; E8640) and the Caspase-9/11 inhibitor Z-LEHD-FMK (Abcam; ab142026) or a DMSO solvent control as the indicated concentrations on the figure legend.

Techniques: Translocation Assay, Activity Assay, Centrifugation, Infection, SDS Page, Western Blot, Staining

Journal: Nature microbiology

Article Title: Spatiotemporal proteomics uncovers cathepsin-dependent macrophage cell death during Salmonella infection

doi: 10.1038/s41564-020-0736-7

Figure Lengend Snippet: a ) Nuclei were extracted from RAW264.7 cells treated with DCG04-Boclicky-TAMRA (5 μM) for 2 hours prior to harvest at 20 hpi with wildtype (Wt), a SPI-2 mutant (Δ ssaV ) or heat killed wildtype bacteria (Wt HK) (MOI = 100:1). Formaldehyde fixed nuclei were subsequently counterstained with Hoechst 33342 and analysed by flow cytometry. Cells in G1, S or G2 phase of the cell cycle are separated by Hoechst 33342 staining on the x-axis. Cathepsin activity (DCG04-Boclicky-TAMRA) is indicated by the % of total DCG04-Boclicky-TAMRA positive nuclei in either sub-G1 or G1/S/G2 or combined total of sub-G1 and G1/2/G2. Data combining all biological replicates from two independent experiments can be seen in Extended Data Fig. 4. b ) Pyroptotic cell death was assessed by quantifying LDH release into culture supernatants of RAW264.7 cells infected with wildtype, SPI-2 (Δ ssaV ) and the effector deletion (Δ sifA) mutants in the presence of 25 μM CA-074-Me or DMSO solvent control at 19 hpi. The Δ sifA mutant has a strong replication defect and readily escapes into the cytoplasm, hyperactivating Caspase-11 dependent cell death . (n) denotes biologically independent samples combined from three independent experiments (batches); each batch contained a minimum of 4 biological replicate wells per condition. Data represents the % LDH release per condition relative maximum LDH release (see Methods). Box plots are depicted as in Fig. 3C. An unpaired t-test (two-sided) was used to calculate p . c ) BMDMs were infected with wild-type S Tm (MOI 100:1), followed by incubation in the presence of cathepsin inhibitor CA-074-Me at the indicated concentrations. At the indicated hpi’s, cell death was measured as the % of LDH released into culture supernatants. Data points represent the mean and error bars indicate the 95% CI. (n) denotes biologically independent samples. Time points 0, 10 and 14 hours are derived from three biological replicates per condition (n=3 per condition) from a single batch, whereas the 18-hour time point contains combined data from 3 or more independent experiments (batches), each batch containing 3-4 biological replicates per condition (DMSO n=30; CA-074-Me (12.5 μM) n=28; CA-074-Me (25 μM) n=13). d ) related to a ), wildtype, caspase-11/1 -/-, caspase-11 -/-, NLRP3 -/-, NLRC4 -/- BMDMs were infected with wildtype S Tm (MOI 100:1), followed by incubation in the presence of cathepsin inhibitor CA-074-Me (12.5 μM) for 18 hpi. The % LDH released into culture supernatants was measured 18 hours post-infection. n denotes biologically independent samples combined from >3 (wildtype) and 3 (mutant genotypes) independent experiments (batches), each batch containing 2-4 biological replicates per condition. A two-sided unpaired t-test was used to calculate p . e ) RAW264.7 cell were transfected with LPS with Fugene (Promega) for 20 hours. Pyroptotic cell death was assessed by quantifying LDH release into culture supernatants in the presence of 25 μM CA-074-Me relative to a DMSO solvent control. (n) denotes biologically independent samples combined from three independent experiments (batches), each batch consisting of a minimum of 3 biological replicate wells per condition. Boxplots are depicted as in Fig. 3C. A two-sided unpaired t-test was used to calculate p .

Article Snippet: In brief, 10 U/100 μL of recombinant Caspase-11 (mouse, Enzo life sciences; BML-SE155-5000) in caspase activity buffer (200 mM NaCl, 50 mM HEPES pH 8.0, 50 mM KCl, 10 mM DTT) supplemented with 100 μMAcLEHD-afc (Santa Cruz; sc-311277) was incubated in the presence of CA-074-Me, E64d (Sigma; E8640) and the Caspase-9/11 inhibitor Z-LEHD-FMK (Abcam; ab142026) or a DMSO solvent control as the indicated concentrations on the figure legend.

Techniques: Mutagenesis, Flow Cytometry, Staining, Activity Assay, Infection, Incubation, Derivative Assay, Transfection

Journal: Nature microbiology

Article Title: Spatiotemporal proteomics uncovers cathepsin-dependent macrophage cell death during Salmonella infection

doi: 10.1038/s41564-020-0736-7

Figure Lengend Snippet: S Tm growth is unaffected by the cathepsin inhibitor CA-074-Me in batch culture conditions. S Tm 14028s growth was measured in the presence of the selective cathepsin inhibitor CA-074-Me in LB 6, 12.5 and 25 μM and DMSO solvent controls. Drug concentrations used are indicated. Experiment was performed in biological triplicate.

Article Snippet: In brief, 10 U/100 μL of recombinant Caspase-11 (mouse, Enzo life sciences; BML-SE155-5000) in caspase activity buffer (200 mM NaCl, 50 mM HEPES pH 8.0, 50 mM KCl, 10 mM DTT) supplemented with 100 μMAcLEHD-afc (Santa Cruz; sc-311277) was incubated in the presence of CA-074-Me, E64d (Sigma; E8640) and the Caspase-9/11 inhibitor Z-LEHD-FMK (Abcam; ab142026) or a DMSO solvent control as the indicated concentrations on the figure legend.

Techniques:

Journal: Nature microbiology

Article Title: Spatiotemporal proteomics uncovers cathepsin-dependent macrophage cell death during Salmonella infection

doi: 10.1038/s41564-020-0736-7

Figure Lengend Snippet: CA-074-Me inhibits cathepsins and reduces expression of proinflammatory proteins. a) 2D-TPP of RAW264.7 cells infected with wildtype S Tm 14028s for 20 hours in the presence of increasing concentrations of CA-074-Me (1-100 μM). To assess CA-074-Me specificity, we applied 2-dimensional Thermal Proteome Profiling (2D-TPP). 2D-TPP enables proteome assessment of protein ligand binding based on the principle that ligand-bound proteins are more thermally stable than proteins not bound to a ligand 20,21 , and in parallel provides information of proteome-wide protein abundance. We subjected RAW264.7 cells infected with wildtype S Tm to increasing doses of CA-074-Me and compared proteome-wide thermostabilisation relative to solvent controlled cells (see methods). Increased (blue) or decreased fold changes are calculated by normalising the abundance of each protein relative to the abundance in the DMSO vehicle per temperature. Protein stabilization (red framed) is demonstrated by increasing (blue) fold changes with respect to increasing temperature (top to bottom) as well as with increasing drug concentrations (left to right). Changes in protein expression (green framed) can be detected if abundance changes are already evident from low temperatures i.e. before proteins melt. All cathepsins detected by 2D-TPP are displayed. CtsA and CtsD are serine and aspartic-acid proteases, respectively, and are therefore not targeted by CA-074-Me and serve as negative controls. Contrary to evidence demonstrating CA-074-Me targets CtsL 35 , we failed to detect its stabilisation in our experiment. Cathepsins are arranged in the top row, followed by caspases and other cellular proteases in descending order. Note Caspase-1 stability was not affected by CA-074-Me, in line with previous reports 22 . Additionally, CA-074-Me reduced IL-1b expression as previously shown 35,36 . 2D-TPP data can be found in . b) Caspase-11-like proteolysis activity was assessed using purified active Caspase-11 and measuring cleavage of the fluorescent substrate AcLEHD-afc as previously described 37 . Caspase-11-like proteolytic activity was assessed in the presence of indicated concentrations of CA-074-Me and the Caspase-11 inhibitor Z-LEHD-FMK as a positive control. Activity is expressed as the % of AcLEHD-afc cleavage relative to the DMSO solvent control. Data is combined from n = 3 biologically independent experiments; error bars depict standard deviation and the center value denotes the mean. c) 2D-TPP profiles of inflammation related proteins reduced in abundance upon CA-074-Me treatment. Data presented as in (a).

Article Snippet: In brief, 10 U/100 μL of recombinant Caspase-11 (mouse, Enzo life sciences; BML-SE155-5000) in caspase activity buffer (200 mM NaCl, 50 mM HEPES pH 8.0, 50 mM KCl, 10 mM DTT) supplemented with 100 μMAcLEHD-afc (Santa Cruz; sc-311277) was incubated in the presence of CA-074-Me, E64d (Sigma; E8640) and the Caspase-9/11 inhibitor Z-LEHD-FMK (Abcam; ab142026) or a DMSO solvent control as the indicated concentrations on the figure legend.

Techniques: Expressing, Infection, Ligand Binding Assay, Activity Assay, Purification, Positive Control, Standard Deviation

Journal: Nature microbiology

Article Title: Spatiotemporal proteomics uncovers cathepsin-dependent macrophage cell death during Salmonella infection

doi: 10.1038/s41564-020-0736-7

Figure Lengend Snippet: Rapid cell death induced by SPI-1 ON S Tm is independent of cathepsin activity. Wildtype and Caspase-1/11 -/- BMDMs were infected with wildtype S Tm and a SPI-1 mutant (Δ prgK ), late-exponential growing S Tm (SPI-1 ON) in the presence of CA-074-Me at the indicated concentrations. LDH release was measured 1.5 hpi. Boxplots are depicted as in . n denotes the number of biologically independent samples.

Article Snippet: In brief, 10 U/100 μL of recombinant Caspase-11 (mouse, Enzo life sciences; BML-SE155-5000) in caspase activity buffer (200 mM NaCl, 50 mM HEPES pH 8.0, 50 mM KCl, 10 mM DTT) supplemented with 100 μMAcLEHD-afc (Santa Cruz; sc-311277) was incubated in the presence of CA-074-Me, E64d (Sigma; E8640) and the Caspase-9/11 inhibitor Z-LEHD-FMK (Abcam; ab142026) or a DMSO solvent control as the indicated concentrations on the figure legend.

Techniques: Activity Assay, Infection, Mutagenesis

Journal: Nature microbiology

Article Title: Spatiotemporal proteomics uncovers cathepsin-dependent macrophage cell death during Salmonella infection

doi: 10.1038/s41564-020-0736-7

Figure Lengend Snippet: CA-074-Me does not affect Caspase-11 expression or processing. Wildtype BMDMs were infected with wildtype S Tm for 20 hours in the presence of CA-074-Me (25 μM) or DMSO control. Lysates were probed for Caspase-11 by immunoblot and using GAPDH as a loading control. Experiment was performed once.

Article Snippet: In brief, 10 U/100 μL of recombinant Caspase-11 (mouse, Enzo life sciences; BML-SE155-5000) in caspase activity buffer (200 mM NaCl, 50 mM HEPES pH 8.0, 50 mM KCl, 10 mM DTT) supplemented with 100 μMAcLEHD-afc (Santa Cruz; sc-311277) was incubated in the presence of CA-074-Me, E64d (Sigma; E8640) and the Caspase-9/11 inhibitor Z-LEHD-FMK (Abcam; ab142026) or a DMSO solvent control as the indicated concentrations on the figure legend.

Techniques: Expressing, Infection, Western Blot

Journal: Nature microbiology

Article Title: Spatiotemporal proteomics uncovers cathepsin-dependent macrophage cell death during Salmonella infection

doi: 10.1038/s41564-020-0736-7

Figure Lengend Snippet: a ) Two-dimensional thermal proteome profiling (2D-TPP) of RAW264.7 cells infected with wildtype S Tm for 20 hours in the presence of increasing concentrations of CA-074-Me post S Tm uptake. Heatmap of Gasdermin D shows decreased abundance (evident from decrease in protein abundance at lower temperatures, before protein melts) with increasing CA-074-Me concentrations. Key is presented to the left of the heatmap; for each protein and temperature, the signal intensity was normalized to the DMSO vehicle control. 2D-TPP data can be found in Supplementary Table 6 and Extended Data Fig. 6. b ) BMDMs were infected with wildtype Salmonella 14028s (20 hpi) and cell lysates analysed by immunoblot for Gasdermin-D and GAPDH as a loading control. A replicate blot from an independent experiment is located in the Supplementary Information (SI). c ) BMDMs infected with wildtype S Tm (MOI = 100:1) were treated with CA-074-Me (12.5 or 25 μM) or DMSO control at 1 or 9 hpi. Box plots are depicted as in Fig. 3C. A two-sided unpaired t-test was used to calculate p . n denotes the combined data from 3 independent experiments (batch), each batch containing 3-4 biological replicates per condition. d ) Schematic depicting Stefin B constructs used to generate iMAC cell lines targeting Stefin B to the nucleus (Nuc) with a 3x NLS sequence or expressed in the cytoplasm (Cyto). Stefin B potently inhibits several cathepsins (e.g. CtsL, CtsS, CtsH and to a lesser extent CtsB) and has been previously used to block cathepsin activity and overcome cathepsin redundancy . Retrovirally transduced iMACs were FACS sorted for low (Lo) and high (Hi) expression of the Stefin B fusion proteins, which was then verified by immunoblot (Extended Data Fig. 10a) as well as localisation of the Stefin B fusion protein by microscopy (Extended Data Fig. 10b). We tested the sensitivity of Stefin B expressing cells to conditions known to activate the canonical inflammasome (i.e. nigericin + LPS) and the lysosome destablising agent, LLoMe, which triggers a distinct form of cell death. In line with our previous observations that cathepsin activity is not required for canonical inflammasome activation, cells with nuclear-expressed Stefin B were refractory to nigericin+LPS treatment, but also to LLoMe induced cell death (Extended Data Fig. 10c, d). e ) iMACs expressing Stefin B targeted to the nucleus, or expressed in the cytoplasm, were infected with wildtype S Tm (MOI = 100:1; 19 hpi). LDH release was quantified as in Fig 5 b . Sample labels are as described in d . Box plots are depicted as in Fig 3 c . A one sided unpaired t-test was used to calculate p . n denotes the combined data from 4 independent experiments (batches), each batch containing >8 biological replicates per condition.

Article Snippet: In brief, 10 U/100 μL of recombinant Caspase-11 (mouse, Enzo life sciences; BML-SE155-5000) in caspase activity buffer (200 mM NaCl, 50 mM HEPES pH 8.0, 50 mM KCl, 10 mM DTT) supplemented with 100 μMAcLEHD-afc (Santa Cruz; sc-311277) was incubated in the presence of CA-074-Me, E64d (Sigma; E8640) and the Caspase-9/11 inhibitor Z-LEHD-FMK (Abcam; ab142026) or a DMSO solvent control as the indicated concentrations on the figure legend.

Techniques: Infection, Western Blot, Construct, Sequencing, Blocking Assay, Activity Assay, Expressing, Microscopy, Activation Assay

Journal: PLoS ONE

Article Title: Regulation of TAK1/TAB1-Mediated IL-1β Signaling by Cytoplasmic PPARβ/δ

doi: 10.1371/journal.pone.0063011

Figure Lengend Snippet: ( A ) Components of cytokine-induced TAK1 signaling and effects of PPARβ/δ identified in the present study. ( B ) Immunoblot analysis of the indicated proteins at different time points after IL-1β stimulation of si-con and si-PPARD treated HeLa cells. P-p65: p65 phosphorylated at serine-536, P-IκB: serine 32; P-p38: threonine-180 and tyrosine-182; P-TAK1: threonine-187. The siRNA effect on PPARβ/δ protein levels in this experiment is shown in . ( C ) Quantification of data obtained by immunoblotting for phosphorylated p65, IκB, p38 and TAK1 as in panel B. Values for phosphoproteins were normalized to signals measured for total protein levels. ( D ) Effect of PPARβ/δ overexpression on IL-1β induced NFκB activity. HeLa cells were transfected with a NFκB-luciferase reporter plasmid and FLAG-PPARβ/δ expression vector or empty vector, and treated with IL-1β for 4 h as indicated. Luciferase activities were determined in cell lysates and normalized to β-galactosidase expressed from a cotransfected CMV-lacZ plasmid. ( E ) Effect of PPARβ/δ overexpression on TAK1/TAB1-induced NFκB activity. Experimental setup as in panel D, expect that TAK1 and TAB1 expression vectors were used instead of IL-1β. ( F ) Effect of PPARβ/δ overexpression on p65-induced NFκB activity. Experimental setup as in panel D, expect that a 65 expression vector was used instead of IL-1β.

Article Snippet: Antibodies against the following proteins or peptides were used for immunoblotting and immunoprecipitation: actin (JLA20; EMD) and TAK1 (sc-7162), TAB1 (sc-13956), p65 NF-kB (sc-372), P(S536)-p65 (sc-3033), IkBa (sc-9242), P(S32)-IkBa (sc-2859) all from Santa Cruz, MYC (9E10), HA (12CA5), GFP (clone 7.1 and 13.1) all from Roche, FLAG M2 (F1804, Sigma), P(T180/Y182)-p38 MAPK (36–850, Invitrogen), and p38 MAPK (raised against ISFVPPPLDQEEMES; rat p38a with C-terminal 15 residues) , TAK1 (4505), P(T187)-TAK1 (4536), TAB1 (C25E9) all from Cell signaling, HSP27 (ADI-SPA-803) from Stressgen, and GFP-Trap_A coupled to agarose beads from Chromotek.

Techniques: Western Blot, Over Expression, Activity Assay, Transfection, Luciferase, Plasmid Preparation, Expressing

Journal: PLoS ONE

Article Title: Regulation of TAK1/TAB1-Mediated IL-1β Signaling by Cytoplasmic PPARβ/δ

doi: 10.1371/journal.pone.0063011

Figure Lengend Snippet: ( A ) Co-immunoprecipitation of PPARβ/δ with TAK1 and TAB1. HEK293IL-1R cells were transfected with expression vectors for MYC-tagged TAB1, GFP-tagged TAK1 and FLAG-tagged PPARβ/δ. Pulldown (PD) was carried out with an antibody against GFP, and immunoblotting with antibodies against TAB1, TAK1 or FLAG. Input lanes were loaded with 50 µg protein (3% of the amount used for IPs). ( B ) Co-immunoprecipitation of PPARβ/δ and TAK1. HEK293IL-1R cells were transfected with expression vectors for HA-tagged TAK1 and/or FLAG-tagged PPARβ/δ. Pulldown (PD) was carried out with an antibody against FLAG, and immunoblotting with antibodies against TAK1 or FLAG. ( C ) Co-immunoprecipitation of PPARβ/δ and TRAF6. HEK293IL-1R cells were transfected with expression vectors for FLAG-tagged TRAF6 and/or CFP-tagged PPARβ/δ. Pulldown (PD) was carried out with an antibody against GFP, and immunoblotting with antibodies against GFP or TRAF6. ( D ) Co-immunoprecipitation of PPARβ/δ and p65. HEK293IL-1R cells were transfected with expression vectors for FLAG-tagged PPARβ/δ and/or HA-tagged p65. Pulldown (PD) was carried out with an antibody against FLAG, and immunoblotting with antibodies against FLAG or HA. ( E ) Cytoplasmic interactions of FLAG-PPARβ/δ with endogenous TAK1. HEK293T cells were transfected with expression vectors for FLAG-tagged PPARβ/δ. Cells were fractionated into cytoplasmic and nuclear fractions, pulldown (PD) was carried out with an antibody against FLAG, and immunoblotting with antibodies against TAK1, FLAG and actin. ( F ) Subcellular localization of endogenous PPARβ/δ in HEK293T cells. Cytoplasmic and nuclear fractions were isolated and analyzed by immunoblotting with an antibody against PPARβ/δ. Antibodies against lactate dehydrogenase (LDH) and acetyl-Histon H3 were included in panels E and F to control for the purity of the cytoplasmic and nuclear fractions.

Article Snippet: Antibodies against the following proteins or peptides were used for immunoblotting and immunoprecipitation: actin (JLA20; EMD) and TAK1 (sc-7162), TAB1 (sc-13956), p65 NF-kB (sc-372), P(S536)-p65 (sc-3033), IkBa (sc-9242), P(S32)-IkBa (sc-2859) all from Santa Cruz, MYC (9E10), HA (12CA5), GFP (clone 7.1 and 13.1) all from Roche, FLAG M2 (F1804, Sigma), P(T180/Y182)-p38 MAPK (36–850, Invitrogen), and p38 MAPK (raised against ISFVPPPLDQEEMES; rat p38a with C-terminal 15 residues) , TAK1 (4505), P(T187)-TAK1 (4536), TAB1 (C25E9) all from Cell signaling, HSP27 (ADI-SPA-803) from Stressgen, and GFP-Trap_A coupled to agarose beads from Chromotek.

Techniques: Immunoprecipitation, Transfection, Expressing, Western Blot, Isolation, Control

Journal: PLoS ONE

Article Title: Regulation of TAK1/TAB1-Mediated IL-1β Signaling by Cytoplasmic PPARβ/δ

doi: 10.1371/journal.pone.0063011

Figure Lengend Snippet: ( A ) Domain structure of PPARβ/δ and TAK1. Deletion mutants were constructed as to preserve or remove specific domains in PPARβ/δ (N-terminal activation domain AF1, DNA-binding domain DBD, ligand binding activation domain LBD/AF2 or the hinge region between the DBD and the LBD/AF2 domains) and in TAK1 (kinase domain). Additional truncations at the C-terminus of TAK-1 mimic known splice variants . ( B ) Interaction of PPARβ/δ deletion mutants with TAK1 and TAB1. Experimental details were as in . ( C ) Effect of PPARβ/δ deletion mutants on TAK1/TAB1 induced NFκB activity. Experimental details were as in . ( D ) Interaction of TAK1 deletion mutants with PPARβ/δ. Experimental details were as in . ( E ) Dependence of the PPARβ/δ enhancement of NFκB activity on catalytically active TAK1. Values represent averages ±SD ( n = 3–5). ***, **, *significant differences between samples as indicated ( p <0.001, p <0.01, p <0.05 by t-test).

Article Snippet: Antibodies against the following proteins or peptides were used for immunoblotting and immunoprecipitation: actin (JLA20; EMD) and TAK1 (sc-7162), TAB1 (sc-13956), p65 NF-kB (sc-372), P(S536)-p65 (sc-3033), IkBa (sc-9242), P(S32)-IkBa (sc-2859) all from Santa Cruz, MYC (9E10), HA (12CA5), GFP (clone 7.1 and 13.1) all from Roche, FLAG M2 (F1804, Sigma), P(T180/Y182)-p38 MAPK (36–850, Invitrogen), and p38 MAPK (raised against ISFVPPPLDQEEMES; rat p38a with C-terminal 15 residues) , TAK1 (4505), P(T187)-TAK1 (4536), TAB1 (C25E9) all from Cell signaling, HSP27 (ADI-SPA-803) from Stressgen, and GFP-Trap_A coupled to agarose beads from Chromotek.

Techniques: Construct, Activation Assay, Binding Assay, Ligand Binding Assay, Activity Assay

Journal: PLoS ONE

Article Title: Regulation of TAK1/TAB1-Mediated IL-1β Signaling by Cytoplasmic PPARβ/δ

doi: 10.1371/journal.pone.0063011

Figure Lengend Snippet: ( A ) Venn Diagram showing the overlaps of genes induced by IL-1β (blue; threshold ≥2-fold; n = 34; n = 113), downregulated by si-HSP27 (yellow; threshold ≥1.5-fold; n = 469) or downregulated by si-PPARD (red; threshold ≥1.8-fold; n = 155). HeLa cells were treated with control siRNA ( si-con) or gene-specific siRNAs followed by IL-1β (10 ng/ml) for 1 h (see and for knockdown efficiency). Expression patterns were determined by microarray analyses and genes showing a ≥1.5-fold regulation were identified . The observed regulation was verified by RT-qPCR, as exemplified for the genes listed in the boxed areas and shown in . ( B, C ) Effect of HSP27 or PPARβ/δ depletion on the IL-1β-mediated induction of the IL6 (B) and IL8 (C) genes in HeLa cells determined by RT-qPCR. Values represent averages ±SD ( n = 3). ***, **, *significant difference between si-con and si-PPARD-treated cells (p<0.001, p <0.01, p <0.05 by t-test). ( D ) Cytoplasmic interaction of PPARβ/δ and HSP27 detected by co-immunoprecipitation. HEK293T cells were transfected with expression vectors for HA-tagged HSP27 and/or FLAG-tagged PPARβ/δ. Cells were fractionated into cytoplasmic and nuclear fractions, pulldown (PD) was carried out with an antibody against FLAG and immunoblotting with anti-HA antibodies. ( E ) Immunoprecipitation of GFP-tagged TAK1 in complexes with FLAG-tagged PPARβ/δ, HA-tagged HSP27. HEK293IL-1R cells were transfected with the indicated expression vectors. Pulldown (PD) was carried out with an antibody against GFP, and immunoblotting with antibodies against TAK1, FLAG or HA. ( F ) Co-expression of PPARβ/δ enhances the interaction of HSP27 and TAK1. HEK293IL-1R cells were transfected with expression vectors for GFP-tagged TAK1, MYC-tagged TAB1, HA-tagged HSP27 and FLAG-tagged PPARβ/δ. Pulldown (PD) was carried out with an antibody against GFP, and immunoblotting with antibodies against TAK1, TAB1, FLAG and HSP27. ( G ) Same experiment as in panel F, except that MYC-TAB1 was omitted. ( H ) Immunoblot analysis of the indicated proteins at different time points after IL-1β stimulation of si-con and si-HSP27 treated HeLa cells. Details as in . The siRNA effect on HSP27 protein levels in this experiment is shown in . ( I ) Effects of siRNA-mediated depletion of PPARβ/δ or/and HSP27 on the IL-1β-induced transcription of IL6 (6 h stimulation with IL-1β). Values represent averages ±SD ( n = 3). ***, **, *significant differences ( p <0.001, p <0.01, p <0.05 by t-test).

Article Snippet: Antibodies against the following proteins or peptides were used for immunoblotting and immunoprecipitation: actin (JLA20; EMD) and TAK1 (sc-7162), TAB1 (sc-13956), p65 NF-kB (sc-372), P(S536)-p65 (sc-3033), IkBa (sc-9242), P(S32)-IkBa (sc-2859) all from Santa Cruz, MYC (9E10), HA (12CA5), GFP (clone 7.1 and 13.1) all from Roche, FLAG M2 (F1804, Sigma), P(T180/Y182)-p38 MAPK (36–850, Invitrogen), and p38 MAPK (raised against ISFVPPPLDQEEMES; rat p38a with C-terminal 15 residues) , TAK1 (4505), P(T187)-TAK1 (4536), TAB1 (C25E9) all from Cell signaling, HSP27 (ADI-SPA-803) from Stressgen, and GFP-Trap_A coupled to agarose beads from Chromotek.

Techniques: Control, Knockdown, Expressing, Microarray, Quantitative RT-PCR, Immunoprecipitation, Transfection, Western Blot

Journal: PLoS ONE

Article Title: Regulation of TAK1/TAB1-Mediated IL-1β Signaling by Cytoplasmic PPARβ/δ

doi: 10.1371/journal.pone.0063011

Figure Lengend Snippet: Untransfected HEK293T cells were treated with formaldehyde to stabilize protein interactions following the protocol for ChIP analyses. Cell extracts were prepared and immuneprecipitations were carried out with either irrelevant IgG or with antibodies against PPARβ/δ. RXR, HSP27, TAK1 or TAB1 (IP). Immunoblotting was performed with PPARβ/δ-specific antibodies. Antibodies against the established PPAR heterodimerization partner RXR were included as a positive control. The PPARβ/δ-HSP27 co-immunoprecipitation was abolished after pretreatment of the cell with HSP27 siRNA, confirming its specificity (not shown). The two rightmost lanes represent untreated extracts from HCT116 cells with intact (+/+) or disrupted (−/−) PPARD alleles to allow for unambiguous identification of the PPARβ/δ band. *, non-specific band.

Article Snippet: Antibodies against the following proteins or peptides were used for immunoblotting and immunoprecipitation: actin (JLA20; EMD) and TAK1 (sc-7162), TAB1 (sc-13956), p65 NF-kB (sc-372), P(S536)-p65 (sc-3033), IkBa (sc-9242), P(S32)-IkBa (sc-2859) all from Santa Cruz, MYC (9E10), HA (12CA5), GFP (clone 7.1 and 13.1) all from Roche, FLAG M2 (F1804, Sigma), P(T180/Y182)-p38 MAPK (36–850, Invitrogen), and p38 MAPK (raised against ISFVPPPLDQEEMES; rat p38a with C-terminal 15 residues) , TAK1 (4505), P(T187)-TAK1 (4536), TAB1 (C25E9) all from Cell signaling, HSP27 (ADI-SPA-803) from Stressgen, and GFP-Trap_A coupled to agarose beads from Chromotek.

Techniques: Western Blot, Positive Control, Immunoprecipitation

Journal: Journal of the Endocrine Society

Article Title: Functional Characterization of Glucocorticoid Receptor Variants Is Required to Avoid Misinterpretation of NGS Data

doi: 10.1210/js.2019-00028

Figure Lengend Snippet: Schematic representation of NR3C1 gene organization and localization of the six missense variants in the NTD of the GR α protein. The NR3C1 gene is located in chromosome 5 in humans and contains at least 10 exons (exon 9 β is not illustrated). The first one is an untranslated exon. Exon 2, the first translated exon, encodes the entire NTD (aa 1–420) composed of the activation function 1 (AF1) domain (aa 77–262), which encompasses the tau core 1 ( τ 1) domain (aa 187–244). Exons 3 and 4 encode the two zinc fingers of the DNA-binding domain (DBD; aa 421–487), whereas exons 5 to 9 encode the hinge region (HR; aa 488–526) and the ligand-binding domain (LBD; aa 527–777) of the GR α protein. All six missense genetic variants (arrows) discovered by NGS are located in the NTD.

Article Snippet: After electroblotting onto C nitrocellulose membranes and incubation with blocking solution [5% fat-free dry milk in Tris-buffered saline containing 1% Tween 20], immunoblotting was performed overnight at 4°C using a mouse monoclonal anti-hGR α antibody directed against the NTD of the protein (SC393232, Santa Cruz Biotechnology ® ) [ ] at a final concentration of 1 μg/mL and with the anti‒ β -actin antibody (Sigma, St Quentin-Falavier, France) [ ] used as a loading control.

Techniques: Activation Assay, Zinc-Fingers, Binding Assay, Ligand Binding Assay

Journal: Journal of the Endocrine Society

Article Title: Functional Characterization of Glucocorticoid Receptor Variants Is Required to Avoid Misinterpretation of NGS Data

doi: 10.1210/js.2019-00028

Figure Lengend Snippet: Protein expression of the WT and the six GR variants. (A) HEK293T cells were transiently transfected or not with a plasmid encoding the WT or the six GR variants. Twenty-four hours posttransfection, protein extracts (20 µg) were analyzed by western blot followed by immunostaining with an antibody recognizing the NTD of GR (29; dilution 1/500) and an anti‒ β -actin antibody (30; dilution 1/1000). GR α protein expression was similar between the WT and the six variants but was below the detectable threshold in the untransfected cells used as control ( i.e., C). Representative image of three independent experiments is shown. The molecular weight marker used was PageRuler TM (Thermo Fisher Scientific). (B) Quantification of the protein signal did not reveal any statistically significant difference between the WT and the GR variants. Results are expressed as means ± SEM.

Article Snippet: After electroblotting onto C nitrocellulose membranes and incubation with blocking solution [5% fat-free dry milk in Tris-buffered saline containing 1% Tween 20], immunoblotting was performed overnight at 4°C using a mouse monoclonal anti-hGR α antibody directed against the NTD of the protein (SC393232, Santa Cruz Biotechnology ® ) [ ] at a final concentration of 1 μg/mL and with the anti‒ β -actin antibody (Sigma, St Quentin-Falavier, France) [ ] used as a loading control.

Techniques: Expressing, Transfection, Plasmid Preparation, Western Blot, Immunostaining, Control, Molecular Weight, Marker