Journal: Physiological Reports

Article Title: Tuberous sclerosis complex exhibits a new renal cystogenic mechanism

doi: 10.14814/phy2.13983

Figure Lengend Snippet: List of antibodies used in the study

Article Snippet: Rabbit polyclonal Anti‐NCC , 1:60 , Stressmarq Biosciences; Victoria, BC, Canada , SPC‐402.

Techniques: Plasmid Preparation, Generated

Journal: Journal of medicinal chemistry

Article Title: Structure-Based Optimization of Small-Molecule Inhibitors for the β -Catenin/B-Cell Lymphoma 9 Protein–Protein Interaction

doi: 10.1021/acs.jmedchem.8b00068

Figure Lengend Snippet: (A) Apparent KD values of wild-type and mutant β-catenin proteins with wild-type BCL9 HD2 domain. (B) AlphaScreen competitive inhibition assay of 3, 27, and 29 to disrupt wild-type β-catenin/wild-type BCL9 and mutant β-catenin/wild-type BCL9 PPIs. Each set of data was expressed as mean ± standard deviation (n = 3). (C) AutoDock docking result of 3 with β-catenin (PDB ID, 2GL7). (D) AutoDock docking result of 29 with β-catenin (PDB ID, 2GL7).

Article Snippet: For both cell lysate and whole cell co-IP experiments, the lysates were then preadsorbed to A/G plus agarose (sc-2003, Santa Cruz Biotechnology) at 4 °C for 1 h. Preadsorbed lysates were incubated with a specific primary antibody against β -catenin (610153, BD Biosciences) overnight at 4 °C.

Techniques: Mutagenesis, Amplified Luminescent Proximity Homogenous Assay, Inhibition, Standard Deviation

Journal: Journal of medicinal chemistry

Article Title: Structure-Based Optimization of Small-Molecule Inhibitors for the β -Catenin/B-Cell Lymphoma 9 Protein–Protein Interaction

doi: 10.1021/acs.jmedchem.8b00068

Figure Lengend Snippet: MTS Assay To Monitor the Inhibitory Activities of 1–32 on the Viability of Cancer Cells, and the Wnt-Responsive TOPFlash Luciferase Reporter Assay To Examine the Effect on Wnt/ β -catenin Transactivation a

Article Snippet: For both cell lysate and whole cell co-IP experiments, the lysates were then preadsorbed to A/G plus agarose (sc-2003, Santa Cruz Biotechnology) at 4 °C for 1 h. Preadsorbed lysates were incubated with a specific primary antibody against β -catenin (610153, BD Biosciences) overnight at 4 °C.

Techniques: MTS Assay, Luciferase, Reporter Assay

Journal: Journal of medicinal chemistry

Article Title: Structure-Based Optimization of Small-Molecule Inhibitors for the β -Catenin/B-Cell Lymphoma 9 Protein–Protein Interaction

doi: 10.1021/acs.jmedchem.8b00068

Figure Lengend Snippet: Wnt-responsive FOPFlash luciferase reporter assay results of inhibitors 1–4, 8, 11, 14, 15, and 29–32 in β-catenin activated HEK293 cells.

Article Snippet: For both cell lysate and whole cell co-IP experiments, the lysates were then preadsorbed to A/G plus agarose (sc-2003, Santa Cruz Biotechnology) at 4 °C for 1 h. Preadsorbed lysates were incubated with a specific primary antibody against β -catenin (610153, BD Biosciences) overnight at 4 °C.

Techniques: Luciferase, Reporter Assay

Journal: Journal of medicinal chemistry

Article Title: Structure-Based Optimization of Small-Molecule Inhibitors for the β -Catenin/B-Cell Lymphoma 9 Protein–Protein Interaction

doi: 10.1021/acs.jmedchem.8b00068

Figure Lengend Snippet: (A,B) Western blot analysis to monitor the change of protein expression of cyclin D1, c-myc, the active form of β-catenin (ABC), and the total β-catenin in response to different concentrations of 3 (A) and 29 (B) in SW480 cells. β-Tubulin was used as the internal reference. The quantitative data of Western blot analyses are shown in Supplementary Table S1. (C) Co-IP experiments to evaluate the disruption of the β-catenin/BCL9 PPI by 29 in HCT116 cell lysate. (D) Co-IP experiments to evaluate the disruption of the β-catenin/BCL9 PPI by 29 and the selectivity of 29 for β-catenin/BCL9 over β-catenin/E-cadherin PPIs in HCT116 cells. IP, immunoprecipitation; IB, immunoblotting; input, 10% of cell lysate. Each experiment was performed in triplicate. The quantitative data of co-IP experiments are shown in Supplementary Table S2.

Article Snippet: For both cell lysate and whole cell co-IP experiments, the lysates were then preadsorbed to A/G plus agarose (sc-2003, Santa Cruz Biotechnology) at 4 °C for 1 h. Preadsorbed lysates were incubated with a specific primary antibody against β -catenin (610153, BD Biosciences) overnight at 4 °C.

Techniques: Western Blot, Expressing, Co-Immunoprecipitation Assay, Immunoprecipitation

Journal: Frontiers in Immunology

Article Title: Critical Role for the NLRP3 Inflammasome in Mediating IL-1β Production in Shigella sonnei -Infected Macrophages

doi: 10.3389/fimmu.2020.01115

Figure Lengend Snippet: S. sonnei induces the secretion of IL-1β, IL-18, NLRP3, ASC, and active caspase-1 in macrophages. (A) J774A.1 macrophages, THP-1 macrophages, PBMCs or BMDM were primed with 1 μg/ml LPS for 4 h and then infected with S. sonnei for an additional 20 h. The levels of IL-1β in the supernatants were measured by ELISA. (B–E) J774A.1 macrophages or THP-1 macrophages were primed with 1 μg/ml LPS for 4 h followed and then infected with S. sonnei for an additional 20 h or stimulated with 5 mM ATP for an additional 0.5 h. The levels of IL-1β (B) , IL-18 (C) , caspase-1 (D) , NLRP3, and ASC (E) in the supernatants were measured by Western blotting. The ELISA data are expressed as the mean ± SD of four separate experiments. The Western blotting results are representative of three different experiments. * and *** indicate significant differences at the levels of p < 0.05 and p < 0.001, respectively, compared to untreated control cells (one-way ANOVA with Dunnett's multiple comparisons test).

Article Snippet: Antibodies against IL-1β (AB-401-NA, polyclonal antibody) were purchased from R&D Systems (Minneapolis, MN).

Techniques: Infection, Enzyme-linked Immunosorbent Assay, Western Blot, Control

Journal: Frontiers in Immunology

Article Title: Critical Role for the NLRP3 Inflammasome in Mediating IL-1β Production in Shigella sonnei -Infected Macrophages

doi: 10.3389/fimmu.2020.01115

Figure Lengend Snippet: S. sonnei induces IL-1β secretion through the NLRP3 inflammasome. (A,B) J774A.1 macrophages or BMDM were primed with 1 μg/ml LPS for 4 h and then treated with YVAD-CHO (A) or MCC950 (B) for 0.5 h. The cells were then infected with 50 MOI S. sonnei for an additional 20 h. The levels of IL-1β in the supernatants were measured by ELISA. (C,D) Wild-type or NLRP3 knockout J774A.1 macrophages were primed with 1 μg/ml LPS for 4 h and then infected with 50 MOI S. sonnei for an additional 20 h. The levels of IL-1β (C) and caspase-1 (D) in the supernatants were measured by ELISA and Western blotting, respectively. The ELISA data are expressed as the mean ± SD of four separate experiments. The Western blotting results are representative of three different experiments. * and *** indicate significant differences at the levels of p < 0.05 and p < 0.001, respectively, compared to S. sonnei -infected cells (A,B) or untreated control cells (C) (one-way ANOVA with Dunnett's multiple comparisons test).

Article Snippet: Antibodies against IL-1β (AB-401-NA, polyclonal antibody) were purchased from R&D Systems (Minneapolis, MN).

Techniques: Infection, Enzyme-linked Immunosorbent Assay, Knock-Out, Western Blot, Control

Journal: Frontiers in Immunology

Article Title: Critical Role for the NLRP3 Inflammasome in Mediating IL-1β Production in Shigella sonnei -Infected Macrophages

doi: 10.3389/fimmu.2020.01115

Figure Lengend Snippet: S. sonnei activates the NLRP3 inflammasome through P 2 X 7 receptor-mediated potassium efflux. (A–C) J774A.1 macrophages were primed with 1 μg/ml LPS for 4 h and then treated with KCl (A) , glibenclamide (B) , probenecid or carbenoxolone (C) for 0.5 h. The cells were then infected with 50 MOI S. sonnei for an additional 20 h. The levels of IL-1β and caspase-1 in the supernatants were measured by ELISA and Western blotting, respectively. (D,E) Mock or P 2 X 7 knockdown J774A.1 macrophages were primed with 1 μg/ml LPS for 4 h and then infected with 50 MOI S. sonnei for an additional 20 h or stimulated with 5 mM ATP for an additional 0.5 h. The levels of IL-1β (D) and caspase-1 (E) in the supernatants were measured by ELISA and Western blotting, respectively. The levels of P 2 X 7 in mock or P 2 X 7 knockdown J774A.1 macrophages were measured by Western blotting and flow cytometry (D) . (F) Mock or P 2 X 7 knockdown J774A.1 macrophages were stimulated with 1 μg/ml LPS for 6 h. The levels of NLRP3 and proIL-1β in the cell lysates were measured by Western blotting. The ELISA data are expressed as the mean ± SD of four separate experiments. The Western blotting results are representative of three different experiments. * and *** indicate significant differences at the levels of p < 0.05 and p < 0.001, respectively, compared to S. sonnei -infected cells (A–C) or untreated control cells (D) (one-way ANOVA with Dunnett's multiple comparisons test).

Article Snippet: Antibodies against IL-1β (AB-401-NA, polyclonal antibody) were purchased from R&D Systems (Minneapolis, MN).

Techniques: Infection, Enzyme-linked Immunosorbent Assay, Western Blot, Knockdown, Flow Cytometry, Control

Journal: Frontiers in Immunology

Article Title: Critical Role for the NLRP3 Inflammasome in Mediating IL-1β Production in Shigella sonnei -Infected Macrophages

doi: 10.3389/fimmu.2020.01115

Figure Lengend Snippet: S. sonnei activates the NLRP3 inflammasome through H 2 O 2 production and lysosomal damage. (A) J774A.1 macrophages were primed with 1 μg/ml LPS for 4 h and then infected with 50 MOI S. sonnei for an additional 20 h. The levels of intracellular ROS were measured by CM-H 2 DCFDA staining, and the data were acquired by flow cytometry. (B) J774A.1 macrophages or BMDM were primed with 1 μg/ml LPS for 4 h and then treated with NAC for 0.5 h. The cells were then infected with 50 MOI S. sonnei for an additional 20 h. The levels of IL-1β and caspase-1 in the supernatants were measured by ELISA and Western blotting, respectively. (C) J774A.1 macrophages or BMDM were primed with 1 μg/ml LPS for 4 h and then treated with NDGA for 0.5 h. The cells were then infected with 50 MOI S. sonnei for an additional 20 h. The levels of IL-1β in the supernatants were measured by ELISA. (D,E) J774A.1 macrophages were primed with 1 μg/ml LPS for 4 h and then treated with NH 4 Cl and CQ (D) or CA-074-Me (E) for 0.5 h. The cells were then infected with 50 MOI S. sonnei for an additional 20 h. The levels of IL-1β in the supernatants were measured by ELISA. The ELISA data are expressed as the mean ± SD of four separate experiments. The flow cytometry and Western blotting results are representative of three different experiments. *, **, and *** indicate significant differences at the levels of p < 0.05, p < 0.01 and p < 0.001, respectively, compared to untreated control cells (A) or S. sonnei -infected cells (B–E) [two-tailed t -test in panel (A) ; one-way ANOVA with Dunnett's multiple comparisons test in panels (B–E) ].

Article Snippet: Antibodies against IL-1β (AB-401-NA, polyclonal antibody) were purchased from R&D Systems (Minneapolis, MN).

Techniques: Infection, Staining, Flow Cytometry, Enzyme-linked Immunosorbent Assay, Western Blot, Control, Two Tailed Test

Journal: Frontiers in Immunology

Article Title: Critical Role for the NLRP3 Inflammasome in Mediating IL-1β Production in Shigella sonnei -Infected Macrophages

doi: 10.3389/fimmu.2020.01115

Figure Lengend Snippet: S. sonnei activates the NLRP3 inflammasome through mitochondrial damage. (A) J774A.1 macrophages were primed with 1 μg/ml LPS for 4 h and then treated with 20 μM MnTBAP for 0.5 h. The cells were then infected with 50 MOI S. sonnei for an additional 20 h. The levels of mitochondrial ROS were measured by MitoSOX staining and flow cytometry. (B) J774A.1 macrophages were primed with 1 μg/ml LPS for 4 h and then treated with MnTBAP for 0.5 h. The cells were then infected with 50 MOI S. sonnei for an additional 20 h. The levels of IL-1β in the supernatants were measured by ELISA. (C) J774A.1 macrophages were primed with 1 μg/ml LPS for 4 h and then infected with 50 MOI S. sonnei for an additional 20 h. The mitochondrial membrane potential was measured by DiOC 2 (3) staining and flow cytometry. (D) J774A.1 macrophages were primed with 1 μg/ml LPS for 4 h and then treated with cyclosporine A for 0.5 h. The cells were then infected with 50 MOI S. sonnei for an additional 20 h. The levels of IL-1β in the supernatants were measured by ELISA. The ELISA data are expressed as the mean ± SD of four separate experiments. The flow cytometry results are representative of three different experiments. ** and *** indicate significant differences at the levels of p < 0.01 and p < 0.001, respectively, compared to S. sonnei -infected cells (B,D) or as indicated (A,C) [two-tailed t -test in panels (A,C) ; one-way ANOVA with Dunnett's multiple comparisons test in panels (B,D) ].

Article Snippet: Antibodies against IL-1β (AB-401-NA, polyclonal antibody) were purchased from R&D Systems (Minneapolis, MN).

Techniques: Infection, Staining, Flow Cytometry, Enzyme-linked Immunosorbent Assay, Membrane, Two Tailed Test

Journal: Frontiers in Immunology

Article Title: Critical Role for the NLRP3 Inflammasome in Mediating IL-1β Production in Shigella sonnei -Infected Macrophages

doi: 10.3389/fimmu.2020.01115

Figure Lengend Snippet: Phagocytosis of live S. sonnei is required for the full activation of the NLRP3 inflammasome. (A) J774A.1 macrophages were primed with 1 μg/ml LPS for 4 h and then treated with cytochalasin D for 0.5 h. The cells were then infected with 50 MOI S. sonnei for an additional 20 h. The levels of IL-1β and TNF-α in the supernatants were measured by ELISA. (B) The TLR2 mRNA expression of mock or TLR2 knockdown J774A.1 macrophages was measured by RT-qPCR. Mock or TLR2 knockdown J774A.1 macrophages were stimulated with 1 μg/ml Pam3CSK4 for 6 h. The levels of TNF-α in the supernatants were measured by ELISA. (C) Mock or TLR2 knockdown J774A.1 macrophages were primed with 1 μg/ml LPS for 4 h and then infected with 50 MOI live, heat-killed or freeze/thaw-killed S. sonnei for an additional 20 h. The levels of IL-1β and TNF-α in the supernatants were measured by ELISA. The ELISA data are expressed as the mean ± SD of four separate experiments. * and *** indicate significant differences at the levels of p < 0.05 and p < 0.001, respectively, compared to S. sonnei -infected cells (A) or live S. sonnei -infected cells (C) or as indicated (B) . ### indicates significant differences at the levels of p < 0.001 compared to sh-SC cells [one-way ANOVA with Dunnett's multiple comparisons test in panels (A,C) ; two-tailed t -test in panel (B) ].

Article Snippet: Antibodies against IL-1β (AB-401-NA, polyclonal antibody) were purchased from R&D Systems (Minneapolis, MN).

Techniques: Activation Assay, Infection, Enzyme-linked Immunosorbent Assay, Expressing, Knockdown, Quantitative RT-PCR, Two Tailed Test

Journal: Frontiers in Immunology

Article Title: Critical Role for the NLRP3 Inflammasome in Mediating IL-1β Production in Shigella sonnei -Infected Macrophages

doi: 10.3389/fimmu.2020.01115

Figure Lengend Snippet: NLRP3 knockout increases the bactericidal activity of macrophages against S. sonnei . (A,B) Wild-type or NLRP3-knockout J774A.1 macrophages were infected with 50 MOI S. sonnei for 15 min or 20 h. The cells were lysed, and the number of engulfed live S. sonnei cells was determined by the CFU assay and is indicated in the upper right corner (A) . The mean CFUs of cells infected with S. sonnei for 15 min or 20 h and the number of killed bacteria were calculated by subtracting the 20 h CFU assay result from the 15 min CFU assay result and are shown in panel (B) . The data are expressed as the mean ± SD of five separate experiments. ***indicates a significant difference at the level of p < 0.001 compared to wild-type cells (two-tailed t- test).

Article Snippet: Antibodies against IL-1β (AB-401-NA, polyclonal antibody) were purchased from R&D Systems (Minneapolis, MN).

Techniques: Knock-Out, Activity Assay, Infection, Colony-forming Unit Assay, Bacteria, Two Tailed Test

Journal:

Article Title: Unilateral Blood Flow Decrease Induces Bilateral and Symmetric Responses in the Immature Brain

doi: 10.2353/ajpath.2009.090257

Figure Lengend Snippet: Unilateral transient CCA occlusion can induce casp3 cleavage but not its downstream subtracts. A: Representative Western blots showing cleaved casp3 fragment (17 kDa) both in the IL and CL 48 hours after uni-tCCAo + MCAo (model M1) and after uni-CCAo (model M2) as compared with sham-operated neocortical cytosolic extracts (n = 8 for each condition). Highly cleaved casp3 was present in the IL after model M1, but it also is increased in the CL cortex and after model M2 compared with sham (sh) cytosolic extracts (P < 0.005). The cytosolic marker β-actin was used as protein loading control. B: Upstream proapoptotic molecules are expressed after neonatal stroke (small units of casp2, casp8, and casp9). After uni-tCCAo, intermediate cleavage fragments of casp2 and cytosolic cytochrome c release were detected, as compared with sham animals. C, upper panel: Western blot showing calpain-cleaved fragments (150 to 145 kDa) and casp3-cleaved fragment (120 kDa) of α II-spectrin, and cleaved poly(ADP-ribose) polymerase-1 fragment (89 kDa). All these fragments were found in the IL extracts after model M1. In contrast, only the 150 kDa fragment was observed after model M2. C, lower panel: Quantification of the 150-kDa band (percentage between OD of the 150-kDa and total 240-kDa α II-spectrin). Data represent mean ± SEM (n = 6 each). *P < 0.05; **P < 0.005; and ***P < 0.0001.

Article Snippet: Antibodies against αII-spectrin (FG 6090; BIOMOL, Coger, Paris, France), poly(ADP-ribose) polymerase-1 (11835238001; Roche), and β-actin (clone AC-15; Sigma-Aldrich) were also used.

Techniques: Western Blot, Marker, Control

Journal: Oncotarget

Article Title: Mouse Sirt3 promotes autophagy in AngII-induced myocardial hypertrophy through the deacetylation of FoxO1

doi: 10.18632/oncotarget.13429

Figure Lengend Snippet: A . Immunoblot analysis of the short form of the Sirt3 was performed in sham and AngII-treated WT and Sirt3-KO mice hearts. Tubulin expression was used as loading control. B . Ratio of the heart weight to body weight in WT and Sirt3-KO mice infused with either saline or AngII for 4 weeks. (n=5) C . The left ventricular wall thickness was measured with echocardiology as described in the methods section. (n=5) D-E . Hematoxylin/eosin stained cardiac sections from control or AngII-treated WT and Sirt3-KO mice showed cardiomyocyte loss or dropout. Masson's trichrome stained sections of the hearts were to detect fibrosis (blue). The graph showed the quantification of interstitial fibrosis in sham or AngII-treated WT and Sirt3-KO mice. (n=5) Scale bar: 20 μm. F . ANF and Myh7 mRNA levels in heart samples of sham or AngII-treated WT and Sirt3-KO mice. (n=5) The data are presented as the means ± SEM of three independent experiments. *P<0.05, **P<0.01.

Article Snippet: Primary antibodies against β-Tubulin (mouse monoclonal) [BM1453], GAPDH (mouse monoclonal) [BM1623], α-SMA (mouse monoclonal) [BM0002] was purchased from Boster.

Techniques: Western Blot, Expressing, Control, Saline, Staining

Journal: Oncotarget

Article Title: Mouse Sirt3 promotes autophagy in AngII-induced myocardial hypertrophy through the deacetylation of FoxO1

doi: 10.18632/oncotarget.13429

Figure Lengend Snippet: A . Immunoblot analysis of Sirt3, LC3 was performed on primary neonatal rat cardioyocytes treated with AngII (1μM, 24h) or chloroquine (CQ, 60μM, 16h). Tubulin expression was used as loading control. B-C . Immunoblot analysis of Sirt3 and autophagic markers was performed on primary neonatal cardioyocytes from WT and Sirt3-KO mice. Tubulin expression was used as loading control. Bar graphs showed the quantification of LC3-II, Beclin-1 and p62 measured by densitometry analysis. (n=5) D-E . The H9C2 cardiomyocytes were transfected with siRNA-Sirt3, and then treated with CQ and AngII. GAPDH expression was used as loading control. Bar graph represents quantification of LC3-II levels measured by densitometry analysis. (n=5) F . The bar graph showing the quantification of ANF and Myh7 mRNA levels as in D. (n=5) The data are presented as the means ± SEM of three independent experiments.*P<0.05, **P<0.01.

Article Snippet: Primary antibodies against β-Tubulin (mouse monoclonal) [BM1453], GAPDH (mouse monoclonal) [BM1623], α-SMA (mouse monoclonal) [BM0002] was purchased from Boster.

Techniques: Western Blot, Expressing, Control, Transfection

Journal: Oncotarget

Article Title: Mouse Sirt3 promotes autophagy in AngII-induced myocardial hypertrophy through the deacetylation of FoxO1

doi: 10.18632/oncotarget.13429

Figure Lengend Snippet: A-B . Immunoblot analysis of FoxO1 and ac-FoxO1 was performed in sham and AngII-treated WT and Sirt3-KO murine hearts. Tubulin expression was used as loading control. Bar graph represents quantification of ac-FoxO1 levels measured by densitometry analysis. (n=5) C-D . Immunoblot analysis of autophagy biomarkers and Sirt3 in the control and siRNA-FoxO1 groups. GAPDH expression was used as loading control. Bar graph represents quantification of LC3-II levels measured by densitometry analysis. (n=5) E . Cellular extracts were immunoprecipitated with Sirt3 antibody and analyzed with anti-FoxO1. F . Cellular extracts were immunoprecipitated with FoxO1 antibody and analyzed with anti-Sirt3. The data are presented as the means ± SEM of three independent experiments.*P<0.05, **P<0.01.

Article Snippet: Primary antibodies against β-Tubulin (mouse monoclonal) [BM1453], GAPDH (mouse monoclonal) [BM1623], α-SMA (mouse monoclonal) [BM0002] was purchased from Boster.

Techniques: Western Blot, Expressing, Control, Immunoprecipitation