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 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: 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

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: Breast cancer research : BCR

Article Title: ERbeta1 represses basal breast cancer epithelial to mesenchymal transition by destabilizing EGFR.

doi: 10.1186/bcr3358

Figure Lengend Snippet: Figure 1 ERb1 inhibits invasion and migration in breast cancer cells by regulating EMT. (A) Control (Lenti), ERa- and ERb1-expressing MDA-MB-231 cells following incubation with EtOH or 17b-estradiol (E2) for 24 h (scale bars, 50 μm). (B) Control (Lenti) and ERb1-expressing Hs578T cells (upper panel) and Hs578T cells that were transiently transfected with a siRNA targeting luciferase (Control) or a specific ERb siRNA (siRNA 3#) (lower panel) were photographed (scale bars, 100 μM). (C) Control (Lenti), ERa- and ERb1-expressing MDA-MB-231 cells were incubated with EtOH or E2 and assessed for invasion by using matrigel-coated Transwell chambers. The cells that were translocated to the lower surface of the filter were shown (left panel) (scale bars, 500 μm). The graph shows the mean (cell number per field) of three separate experiments with the standard error of the mean (SEM) and P-value (*) ≤0.05% indicated. (D) Control (Lenti) and ERb1-expressing MDA-MB-231 cells were incubated with E2 for 24 h and assessed for migration using wound-healing assay. The bar graph shows the mean (cells migrated into the wound) of three separate experiments with SEM and P-value (*) ≤0.05% indicated. (E) E-cadherin protein levels in control (Lenti), ERa- or ERb1-expressing MDA-MB-231 cells. (F) E-cadherin expression was analyzed by immunoblotting in MDA-MB-231 cells transfected with control or ERb siRNA (3#) (upper panel) and qPCR in MDA-MB-231 cells transfected with control or three specific ERb siRNAs (lower panel). The graph indicates the mean of three separate experiments with SEM and P-value (*) ≤0.05%. (G) E-cadherin was visualized by immunofluorescence in control (Lenti) and ERb1-expressing cells (scale bars, 20 μm).

Article Snippet: Primary antibodies used in immunoblotting include: ERa, E-cadherin, N-cadherin, cadherin-11, vimentin, ZEB-1, SIP1, Lamin A/C and Tubulin (Santa Cruz Biotechnology, Santa Cruz, CA USA), actin-b (Sigma St. Louis, MO USA), EGFR (Santa Cruz Biotechnology, Cell Signaling, Danvers, MA USA), ERb1 (14C8; GeneTex, Irvine, CA USA), which detects an N-terminal epitope and recognizes the ERb isoforms derived from alternative splicing of the last exon, including ERb1 and an in-house antibody that detects an epitope in ligand binding domain of ERb1 (amino acids 320 to 527)) [29], SNAIL (Abcam, Cambridge, MA USA), p-ERK1/2 and total ERK1/2 (Cell Signaling), c-Cbl (BD Biosciences).

Techniques: Migration, Control, Expressing, Incubation, Transfection, Luciferase, Wound Healing Assay, Western Blot, Immunofluorescence

Journal: Breast cancer research : BCR

Article Title: ERbeta1 represses basal breast cancer epithelial to mesenchymal transition by destabilizing EGFR.

doi: 10.1186/bcr3358

Figure Lengend Snippet: Figure 2 ERb1 induces the expression of E-cadherin by up-regulating members of the microRNA 200 family and repressing the expression of ZEB-1 and SIP-1. (A) E-cadherin mRNA levels in control (Lenti), ERa- and ERb1-expressing MDA-MB-231 cells following incubation with or without E2 for 24 h. The graph shows the mean of three separate experiments with SEM and P-value (*) ≤0.05% indicated. (B) Left panel: protein levels of EMT markers in control (Lenti), ERa- and ERb1-expressing MDA-MB-231 cells following incubation with or without E2 for 24 h. Right panel: E- and N-cadherin protein levels in control (Lenti) and ERb1-expressing Hs578T cells. (C) ZEB-1 and SIP-1 protein levels in control (Lenti), ERa- and ERb1-expressing MDA-MB-231 cells (upper panel) and in control and ERb1-expressing Hs578T cells (lower panel). (D) Control, ERa- and ERb1-expressing MDA-MB-231 cells were analyzed for miR-200a, miR-200b and miR-429 expression by qPCR. The graphs show data as fold change compared with the untreated Lenti cells (mean of three separate experiments with SEM and P-value (*) ≤0.05% indicated). (E) MDA-MB-231 cells were transiently transfected with control or ERb siRNA (3#) and analyzed for miR-200a, miR-200b and miR-429 expression by qPCR. The graphs show the mean of three separate experiments with SEM and P-value (*) ≤0.05% indicated. (F) ERb1-expressing MDA-MB-231 cells were transfected with inhibitors of miR-200a, miR-200b and miR-429 or a negative control inhibitor. Cells were photographed and analyzed for E-cadherin expression by qPCR (scale bars, 50 μm). The level of functional knockdown of miR-200a-b-429 was examined by a miR-200a-b-429-regulated reporter assay. The graphs show the mean of three separate experiments with SEM and P-value (*) ≤0.05% indicated.

Article Snippet: Primary antibodies used in immunoblotting include: ERa, E-cadherin, N-cadherin, cadherin-11, vimentin, ZEB-1, SIP1, Lamin A/C and Tubulin (Santa Cruz Biotechnology, Santa Cruz, CA USA), actin-b (Sigma St. Louis, MO USA), EGFR (Santa Cruz Biotechnology, Cell Signaling, Danvers, MA USA), ERb1 (14C8; GeneTex, Irvine, CA USA), which detects an N-terminal epitope and recognizes the ERb isoforms derived from alternative splicing of the last exon, including ERb1 and an in-house antibody that detects an epitope in ligand binding domain of ERb1 (amino acids 320 to 527)) [29], SNAIL (Abcam, Cambridge, MA USA), p-ERK1/2 and total ERK1/2 (Cell Signaling), c-Cbl (BD Biosciences).

Techniques: Expressing, Control, Incubation, Transfection, Negative Control, Functional Assay, Knockdown, Reporter Assay

Journal: Breast cancer research : BCR

Article Title: ERbeta1 represses basal breast cancer epithelial to mesenchymal transition by destabilizing EGFR.

doi: 10.1186/bcr3358

Figure Lengend Snippet: Figure 3 EGFR promotes EMT and its down-regulation is involved in ERb1-induced E-cadherin expression. (A) EGFR, total ERK1/2 and phospho-ERK1/2 levels in control (Lenti), ERa- and ERb1-expressing MDA-MB-231 cells following incubation with or without E2 for 24 h. (B) EGFR protein levels in control (Lenti) and ERb1-expressing Hs578T cells. (C) EGFR protein levels in MDA-MB-231 cells transiently transfected with control or ERb siRNA (3#). (D) ERb1-expressing MDA-MB-231 cells were incubated in absence or presence of 5 ng/ml TGF-b1 or 10 ng/ml EGF for 24 h and photographed (scale bars, 50 μm). (E) ERb1-expressing MDA-MB-231 cells were incubated in absence or presence of 10 ng/ml EGF for 24 h and analyzed for the expression of EGFR, total ERK1/2 and phospho-ERK1/2 by immunoblotting. Note that the decreased EGFR levels following EGF treatment is due to increased degradation. (F) miR-200a, miR-200b and miR-429 levels in control (Lenti) and ERb1-expressing MDA-MB-231 cells following incubation with 5 ng/ml TGF-b1 or 10 ng/ml EGF for 24 h. The graph shows the data as fold change compared with the untreated Lenti cells (mean of three separate experiments (± SEM) with P-value (*) ≤0.05%). (G) E-cadherin mRNA and protein levels in ERb1-expressing MDA-MB-231 cells following incubation with 5 ng/ml TGF-b1 or 10 ng/ml EGF for 24 h. The graph shows the mean of three separate experiments with SEM and P-value (*) ≤0.05% indicated. (H) ERb1-expressing MDA-MB-231 cells were stably co-transfected with an empty pBABE vector (ERb1:pBABE cells) or the pBABE-EGFR plasmid (ERb1:EGFR cells), photographed and analyzed for EGFR, E-cadherin and ERb1 expression by immunoblotting (scale bars, 50 μm).

Article Snippet: Primary antibodies used in immunoblotting include: ERa, E-cadherin, N-cadherin, cadherin-11, vimentin, ZEB-1, SIP1, Lamin A/C and Tubulin (Santa Cruz Biotechnology, Santa Cruz, CA USA), actin-b (Sigma St. Louis, MO USA), EGFR (Santa Cruz Biotechnology, Cell Signaling, Danvers, MA USA), ERb1 (14C8; GeneTex, Irvine, CA USA), which detects an N-terminal epitope and recognizes the ERb isoforms derived from alternative splicing of the last exon, including ERb1 and an in-house antibody that detects an epitope in ligand binding domain of ERb1 (amino acids 320 to 527)) [29], SNAIL (Abcam, Cambridge, MA USA), p-ERK1/2 and total ERK1/2 (Cell Signaling), c-Cbl (BD Biosciences).

Techniques: Expressing, Control, Incubation, Transfection, Western Blot, Stable Transfection, Plasmid Preparation

Journal: Breast cancer research : BCR

Article Title: ERbeta1 represses basal breast cancer epithelial to mesenchymal transition by destabilizing EGFR.

doi: 10.1186/bcr3358

Figure Lengend Snippet: Figure 6 ERb1 levels positively correlate with E-cadherin in breast cancers. (A) Pearson’s correlation of ERb1 expression with expression of E-cadherin. N equals the number of patients for whom data were available. (B) Representative images of ERb1 and E-cadherin expression in two serial sections of the same tumor from two cases. Scale bars represent 200 μM. (C) ERb1 and E-cadherin were box-plotted in the 208 breast cancer patients. The patients were divided into three groups based on ERb1 expression scores in the tumors, representing low, medium and high expression of ERb1. Any outliers were marked with a circle and extreme cases with an asterisk. Data were analyzed using one-way ANOVA test with Games-Howell’s correction. (D) The percentage of E-cadherin-positive tumors was analyzed in the three groups of patients as described in C. Data were analyzed using Pearson’s c2 test. (E) Proposed mechanism for how ERb1 regulates EMT and influences invasion in breast cancer. EGFR promotes EMT in basal cells by activating ERK1/2, which in turn, by inducing the expression of ZEB1/2, results in the down-regulation of E- cadherin. This process requires repression of the expression of members of miR-200 family. By inducing the degradation of EGFR, ERb1 sustains ERK1/2 inactive, up-regulates miR200a-b and miR-429, down-regulates ZEB1/2 and induces the expression of E-cadherin.

Article Snippet: Primary antibodies used in immunoblotting include: ERa, E-cadherin, N-cadherin, cadherin-11, vimentin, ZEB-1, SIP1, Lamin A/C and Tubulin (Santa Cruz Biotechnology, Santa Cruz, CA USA), actin-b (Sigma St. Louis, MO USA), EGFR (Santa Cruz Biotechnology, Cell Signaling, Danvers, MA USA), ERb1 (14C8; GeneTex, Irvine, CA USA), which detects an N-terminal epitope and recognizes the ERb isoforms derived from alternative splicing of the last exon, including ERb1 and an in-house antibody that detects an epitope in ligand binding domain of ERb1 (amino acids 320 to 527)) [29], SNAIL (Abcam, Cambridge, MA USA), p-ERK1/2 and total ERK1/2 (Cell Signaling), c-Cbl (BD Biosciences).

Techniques: Expressing

Journal: The Journal of Biological Chemistry

Article Title: Fibroblast growth factor receptor 5 (FGFR5) is a co-receptor for FGFR1 that is up-regulated in beta-cells by cytokine-induced inflammation

doi: 10.1074/jbc.RA118.003036

Figure Lengend Snippet: FGFR5 exists as preformed higher aggregates at the plasma membrane. A, FGFR5 exhibits extracellular ligand-binding Ig loops similar in structure to canonical FGFRs and a unique truncated noncatalytic C terminus. HA-tagged full-length FGFR5 (R5HA) or C terminus–deficient FGFR5 (ΔCHA) were co-expressed in AD293 cells at a 1:1 molar ratio with Venus-tagged constructs (R5Ven or ΔCVen) in either a homogeneous (B) or heterogeneous (C) pattern. Cultures were stimulated in the absence (−) or presence (+) of FGF2 (10 ng/ml; supplemented with heparin sulfate; 10 min). FGFR5 immunoprecipitated (IP) with variant isoforms of itself, as detected by Western immunoblotting (IB). The ability of FGFR5-ΔC to pull down FGFR5 indicates that the unique intracellular domain is not required for this molecular association. D, a panel of representative images showing 2-photon fluorescence intensity (2P Fluor.) and anisotropy image maps of AD293 cells expressing Venus monomer (Ven. Mon.), tandem Venus dimer (Ven. Dim.), R5Ven, ΔCVen, and R5Ven co-expressed with untagged FGFR5-ΔC (ΔCDark). Changes in anisotropy between the Venus monomer and dimer controls illustrate the independence of this measurement to fluorescence intensity. Scale bar, 10 μm. E, the average anisotropy of each sample in the absence (−) and presence (+) of FGF2 reveals that R5Ven forms a higher-order (n > 2) oligomer that further aggregates with FGF2 stimulation. Truncation of the intracellular domain (ΔCVen) presents a receptor with an average anisotropy value closer to the Venus dimer, independent of FGF2 stimulation. Values from n = 61–165 cells analyzed and pooled from four independent experiments performed on different days are represented as box–whisker plots. Boxes delineate the 25th and 75th percentiles, and center lines indicate the medians. Whiskers extend 1.5 times the interquartile range from the 25th and 75th percentiles. ****. p < 0.0001 based on one-way ANOVA.

Article Snippet: The medium was replaced with fresh culture medium containing cytokines (50 units/ml IL-1β, 750 units/ml IFN-γ, and 1000 units/ml TNFα) and/or 10 ng/ml recombinant human FGF2 (R&D Systems; supplemented with 10 units/ml heparin sodium salt (Sigma-Aldrich)) where indicated for an additional 24 h. Positive control cells were treated with 10 m m H 2 O 2 for 6 h before staining while negative control cells were untreated.

Techniques: Clinical Proteomics, Membrane, Ligand Binding Assay, Construct, Immunoprecipitation, Variant Assay, Western Blot, Fluorescence, Expressing, Whisker Assay

Journal: The Journal of Biological Chemistry

Article Title: Fibroblast growth factor receptor 5 (FGFR5) is a co-receptor for FGFR1 that is up-regulated in beta-cells by cytokine-induced inflammation

doi: 10.1074/jbc.RA118.003036

Figure Lengend Snippet: Anisotropy enhancement curves suggest that FGFR5 forms trimers. A, progressively increasing fluorescence labeling of randomly distributed populations of monomers, dimers, and trimers exhibit differences in the apparent aggregate state. These changes are reflected by the relationship between anisotropy and fluorescence labeling, where monomers (top third of bottom panel; blue curve) appear zero-order, dimers (middle third of bottom panel; green curve) appear first-order (or linear), and trimers (bottom third of bottom panel; red curve) appear second-order. B, the anisotropy of R5Ven increased when co-expressed with increasing amounts of R5Dark (i.e. untagged R5), reflecting a decrease in the fluorescence-labeled fraction. The line of best fit is shown in red. Values from n = 87–162 cells analyzed from three independent experiments performed on different days are represented as box–whisker plots. C, enhancement curves constructed with anisotropy measurements from time series images of AD293 cells expressing Venus monomer and Venus dimer controls that were progressively photobleached after each image acquisition. The slope of the line of best fit for Venus monomer was relatively flat, whereas Venus dimer exhibited a first-order relationship. D, anisotropy enhancement curves for R5Ven exhibited a curve of best fit that approximated a trimeric oligomerization state (2.73 ± 0.05 monomers/aggregate), whereas R5-ΔCVen is best approximated as a dimeric state (2.05 ± 0.05 monomers/aggregate). E, the R5Ven enhancement curve profile is similar in the absence (blue) or presence (green) of FGF2. Enhancement curve data were constructed by analyzing between 18 and 95 cells through 11 frames within a photobleach sequence from three independent experiments performed on different days. F, relative brightness values for Venus-tagged constructs expressed in the absence and presence of Cerulean-tagged constructs suggest that R5Ven is a trimer (blue) and ΔCVen is a dimer (red) and that co-expression of ΔCCer forces R5Ven to undergo a trimer-to-dimer (3 to 2) transition (green). The relative brightness of R5Ven expressed alone in the presence versus absence of FGF2 (gray) revealed no change in the aggregation state. Values from n = 53–163 cells/experiment are represented as box–whisker plots. Boxes delineate the 25th and 75th percentiles, and center lines indicate the medians. Whiskers extend 1.5 times the interquartile range from the 25th and 75th percentiles. *** and ****, p < 0.001 and p < 0.0001, respectively, based on one-way ANOVA. G, model depicting a mixture of FGFR5 homotrimers and homodimers on the plasma membrane. The homotrimer is formed by both C-terminal and extracellular/transmembrane interaction, whereas the stable homodimer is due to C-terminal interaction.

Article Snippet: The medium was replaced with fresh culture medium containing cytokines (50 units/ml IL-1β, 750 units/ml IFN-γ, and 1000 units/ml TNFα) and/or 10 ng/ml recombinant human FGF2 (R&D Systems; supplemented with 10 units/ml heparin sodium salt (Sigma-Aldrich)) where indicated for an additional 24 h. Positive control cells were treated with 10 m m H 2 O 2 for 6 h before staining while negative control cells were untreated.

Techniques: Fluorescence, Labeling, Whisker Assay, Construct, Expressing, Sequencing, Clinical Proteomics, Membrane

Journal: The Journal of Biological Chemistry

Article Title: Fibroblast growth factor receptor 5 (FGFR5) is a co-receptor for FGFR1 that is up-regulated in beta-cells by cytokine-induced inflammation

doi: 10.1074/jbc.RA118.003036

Figure Lengend Snippet: FGFR5 forms a heterocomplex with FGFR1 that responds to FGF2 ligand stimulation. A, left blots, HA-tagged R5 (R5HA) was co-expressed with Venus-tagged R5 (R5Ven) or R1 (R1Ven). Right blots, HA-tagged R1 (R1HA) was co-expressed with R5Ven. Cells were stimulated in the absence (−) or presence (+) of FGF2 (10 ng/ml; supplemented with heparin sulfate; 10 min). Co-immunoprecipitation (IP) with anti-HA antibodies and Western immunoblotting (IB) for the fluorescent protein (FP) reveal an association between FGFR5 and FGFR1. No changes were observed in the presence of ligand stimulation by this method. B, immunoprecipitation with anti-FGFR1 antibody and Western immunoblotting for FGFR5 reveal an association between endogenous FGFR5 and FGFR1. C, anisotropy values of AD293 cells expressing R5Ven and R1Dark at a 1:2 ratio in the absence (blue) and presence (red) of FGF2. The anisotropy of Venus monomer is indicated as a black line, and R5Ven is indicated as gray box–whisker plots as previously presented in Fig. 2E. Values from n = 59–67 cells analyzed from three independent experiments performed on different days are represented as box–whisker plots. **** (gray), p < 0.0001 between R5Ven (gray) and R1Cer in the absence (blue) or presence (red) of FGF2; n.s., indicates no significant difference between the absence (blue) and presence (red) of R5Ven + R1Cer by one-way ANOVA. D, anisotropy enhancement curve produced by progressive photobleaching of AD293 cells expressing R5Ven + R1Dark (red) overlaid on an enhancement curve from cells expressing R5Ven (gray; previously represented in Fig. 3D) reveals a trimer-to-dimer transition upon co-expression of R1Dark. E, the relative brightness comparing R5Ven in the absence and presence of Cerulean-tagged FGFR1 (R1Cer) also suggests a trimer-to-dimer (3 to 2) transition. The relative brightness of cells co-expressing R5Ven with R1Cer in the presence versus absence of FGF2 suggests a doubling of the aggregate. Values from n = 167–213 cells analyzed from five independent experiments performed on different days are represented as box–whisker plots. F, relative brightness comparing R1Ven in the absence versus presence of either R1Cer or R5Cer indicates that FGFR5 co-expression forces a dimer-to-monomer transition (2 to 1) of FGFR1 aggregates. Values from n = 78–92 cells analyzed from four independent experiments performed on different days are represented as box–whisker plots. Boxes delineate the 25th and 75th percentiles, and center lines indicate the medians. Whiskers extend 1.5 times the interquartile range from the 25th and 75th percentiles. G, model depicting FGFR5 and FGFR1 interaction at the plasma membrane. FGFR5 and FGFR1 form a 2:1 heterocomplex ((FGFR5)2/(FGFR1)1) that further aggregates to a 4:2 heterocomplex upon stimulation with FGF2 ((FGFR5)4/(FGFR1)2). The inability to detect FGF2-induced aggregation by FGFR5 homoFRET but observation by N&B analysis of both FGFR5 and FGFR1 suggests that FGFR5 homodimers are on the opposite side of the complex ((FGFR5)2/(FGFR1)2/(FGFR5)2), separated by >4.95 nm.

Article Snippet: The medium was replaced with fresh culture medium containing cytokines (50 units/ml IL-1β, 750 units/ml IFN-γ, and 1000 units/ml TNFα) and/or 10 ng/ml recombinant human FGF2 (R&D Systems; supplemented with 10 units/ml heparin sodium salt (Sigma-Aldrich)) where indicated for an additional 24 h. Positive control cells were treated with 10 m m H 2 O 2 for 6 h before staining while negative control cells were untreated.

Techniques: Immunoprecipitation, Western Blot, Expressing, Whisker Assay, Produced, Clinical Proteomics, Membrane