Journal: Journal of Biological Chemistry

Article Title: Neuropilin-1 Mediates Divergent R-Smad Signaling and the Myofibroblast Phenotype

doi: 10.1074/jbc.m110.151696

Figure Lengend Snippet: FIGURE 1. Smad protein phosphorylation by TGF-1 stimulation in LX2, PSC, and MEF cells. LX2, PSC, and MEF cells were serum-starved overnight, then treated with different concentrations of TGF-1 for 45 min. Antibodies against p-Smad1/5 (Ser-463/465), p-Smad2 (Ser-465/467), and p-Smad3 (Ser-423/425) were used. Both Smad1/5 and Smad2/3 were phosphorylated by TGF-1 stimulation in all of the cells. An asterisk (*) indicates the nonspecific band.

Article Snippet: Antibodies and Other Reagents—NRP-1, Id-1, T RII, and -actin were from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA); Smad2, Smad5, p-Smad1/5, p-Smad2, p-Smad3 were purchased from Cell Signaling Technology, Inc. (Danvers, MA); -SMA was from Millipore (Billerica, MA); collagen I antibody was from Rockland Immunochemicals, Inc. (Gilbertsville, PA); PAI were fromNovus Bio. (Littleton, CO) TGF- 1 was from Biolegend (San Diego, CA); BMP9 was from R&D Systems (Minneapolis, MN); BMP2 and BMP4 were from Senway Biotech (San Diego,CA)ALK5 inhibitors SB431542 andALK5 inhibitors (2-(3- (6-methylpyridine-2-yl)-1H-pyrazol-4-yl)-1,5-naphthyridine) were from Stemgent (Cambridge, MA). siRNA and shRNA Transfection—siRNA for human NRP-1, T RII, and control were from Qiagen, Inc. (Valencia, CA). siRNA transfection was performed by Hiperfect (Qiagen) following the manufacturer’s instructions. shRNA for human NRP-1 and controls were from Open Biosystems (Huntsville, AL) and were prepared as previously described (18).

Techniques: Phospho-proteomics

Journal: Journal of Biological Chemistry

Article Title: Neuropilin-1 Mediates Divergent R-Smad Signaling and the Myofibroblast Phenotype

doi: 10.1074/jbc.m110.151696

Figure Lengend Snippet: FIGURE 2. The effects of eliminating or overexpressing NRP-1 on Smad protein phosphorylation and the Smad target gene expression. A, eliminating NRP-1 up-regulated Smad1/5 phosphorylation and down-regulated Smad2/3 phosphorylation. NRP-1 was knocked down by siRNA in LX2 and PSC. For MEF cells, wt MEF, and NRP-1/ MEF cells were used. All the cells were serum-starved overnight, and then treated with 10 ng/ml TGF-1 for the indicated time. An asterisk (*) indicates the nonspecific band. B, overexpress NRP-1 in NRP-1/ MEF cells down-regulated Smad1/5 phosphorylation and up-regulated Smad2/3 phosphorylation. NRP-1/ MEF cells were infected with NRP-1 encoding retrovirus. LacZ retrovirus was used as control. After 36 h, cells were serum-starved overnight, and then treated with 10 ng/ml TGF-1 for the indicated time. C–E, effect of knocking down NRP-1 on the Smad proteins transcriptional activity in the cells measured by the luciferase promoter assay. 5 103/well LX2 cells were plated into a 96-well plate transfected with NRP-1 siRNA for 24–30 h, then serum-starved overnight. The cells were then transfected with the corresponding luciferase promoter plasmids together with pRL-TKRenilla luciferase plasmid astheinternalcontrol.Onehourafterthetransfection,TGF-1wasaddedtothecorrespondingwelltothefinalconcentrationof10ng/ml.Fireflyluciferaseand Renilla luciferase activities were performed. C, Luc-Id-1 promoter. D, Luc-ARE, co-transfected with FAST. E, Luc-SBE. F, knocking down NRP-1 affected the Smad protein responding gene expression. Cells were transfected with NRP-1 siRNA for 24–30 h in the complete medium, then serum-starved overnight. 10 ng/ml TGF-1 was added to the cells for 24 h. In response to TGF-1 stimulation, Id-1 protein (the Smad1-responding gene) expression was more highly up-regulated in NRP-1 knocked down cells than in control cells stimulated with TGF-1. In response to TGF-1 stimulation, -SMA and PAI-1 protein (the Smad3-responding gene) expression was more highly down-regulated in NRP-1 knocked down cells than in control cells stimulated with TGF-1.

Article Snippet: Antibodies and Other Reagents—NRP-1, Id-1, T RII, and -actin were from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA); Smad2, Smad5, p-Smad1/5, p-Smad2, p-Smad3 were purchased from Cell Signaling Technology, Inc. (Danvers, MA); -SMA was from Millipore (Billerica, MA); collagen I antibody was from Rockland Immunochemicals, Inc. (Gilbertsville, PA); PAI were fromNovus Bio. (Littleton, CO) TGF- 1 was from Biolegend (San Diego, CA); BMP9 was from R&D Systems (Minneapolis, MN); BMP2 and BMP4 were from Senway Biotech (San Diego,CA)ALK5 inhibitors SB431542 andALK5 inhibitors (2-(3- (6-methylpyridine-2-yl)-1H-pyrazol-4-yl)-1,5-naphthyridine) were from Stemgent (Cambridge, MA). siRNA and shRNA Transfection—siRNA for human NRP-1, T RII, and control were from Qiagen, Inc. (Valencia, CA). siRNA transfection was performed by Hiperfect (Qiagen) following the manufacturer’s instructions. shRNA for human NRP-1 and controls were from Open Biosystems (Huntsville, AL) and were prepared as previously described (18).

Techniques: Phospho-proteomics, Targeted Gene Expression, Infection, Control, Activity Assay, Luciferase, Promoter Assay, Transfection, Plasmid Preparation, Gene Expression

Journal: Journal of Biological Chemistry

Article Title: Neuropilin-1 Mediates Divergent R-Smad Signaling and the Myofibroblast Phenotype

doi: 10.1074/jbc.m110.151696

Figure Lengend Snippet: FIGURE 3. NRP-1 interacted with TGF- receptors. A, NRP-1 bound to TGF-RII. LX2 cells grown in the complete medium were made for cell lysate, and immunoprecipitation was performed using anti-NRP-1 antibody and West- ernblotforTGF-RII.ControlantibodywithcelllysateandNRP-1antibodywithoutcelllysatewereusedasnegative controls.B,TGF-1stimulationdidnotincreasetheassociationbetweenNRP-1andTGF-RII.LX2cellswereserum- starved overnight and stimulated with 10 ng/ml TGF-1 for 0, 15, and 45 min. The cell lysate was made, and immu- noprecipitation was performed using anti-NRP-1 antibody and Western blot for TGF-RII. C, siRNA knocked down TGF-RII eliminated the effect of NRP-1 on Smad protein phosphorylation. LX2 cells were transfected with control, NRP-1, and TGF-RII siRNA alone or combined, as indicated in the figure. 24–30 h later, cells were serum-starved overnight. 10 ng/ml TGF-1 was added to the indicated plates for 45 min, and the cell lysate was subjected to Western blot analysis. An asterisk (*) indicates the nonspecific band. D, TGF-RI (ALK5) inhibitors eliminated both Smad1/5 and Smad2/3 protein phosphorylation on TGF-1 stimulation. LX2 cells were serum-starved overnight. TGF-RI inhibitors SB431542 (10 M) and ALK5 Inhibitor (10 M) were added to the cells 30 min before TGF-1 stimulation. An asterisk (*) indicates the nonspecific band. E, NRP-1 associated with TGF-RI (ALK5) by TGF-1 stimulation. 293T cells were transfected with NRP-1 and Flag-ALK5-expressing plasmids. Cells were serum-starved overnight and stimulated with 10 ng/ml TGF-1 for 0, 5, and 15 min. The cell lysate was made, and immunoprecipi- tation was performed using anti-NRP-1 antibody and Western blot for Flag tag. F, knockdown TGF-RII eliminated theassociationbetweenNRP-1andTGF-RI(ALK5)inthepresentofTGF-stimulation.293Tcellsweretransfected with TGF-RII siRNA, and the following experiments were preformed as described in panel E.

Article Snippet: Antibodies and Other Reagents—NRP-1, Id-1, T RII, and -actin were from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA); Smad2, Smad5, p-Smad1/5, p-Smad2, p-Smad3 were purchased from Cell Signaling Technology, Inc. (Danvers, MA); -SMA was from Millipore (Billerica, MA); collagen I antibody was from Rockland Immunochemicals, Inc. (Gilbertsville, PA); PAI were fromNovus Bio. (Littleton, CO) TGF- 1 was from Biolegend (San Diego, CA); BMP9 was from R&D Systems (Minneapolis, MN); BMP2 and BMP4 were from Senway Biotech (San Diego,CA)ALK5 inhibitors SB431542 andALK5 inhibitors (2-(3- (6-methylpyridine-2-yl)-1H-pyrazol-4-yl)-1,5-naphthyridine) were from Stemgent (Cambridge, MA). siRNA and shRNA Transfection—siRNA for human NRP-1, T RII, and control were from Qiagen, Inc. (Valencia, CA). siRNA transfection was performed by Hiperfect (Qiagen) following the manufacturer’s instructions. shRNA for human NRP-1 and controls were from Open Biosystems (Huntsville, AL) and were prepared as previously described (18).

Techniques: Immunoprecipitation, Western Blot, Phospho-proteomics, Transfection, Control, Expressing, FLAG-tag, Knockdown

Journal: Journal of Biological Chemistry

Article Title: Neuropilin-1 Mediates Divergent R-Smad Signaling and the Myofibroblast Phenotype

doi: 10.1074/jbc.m110.151696

Figure Lengend Snippet: FIGURE 8. The schematic illustration of NRP-1 function in regulating TGF- signaling. TGF- induced both Smad1/5/8 and Smad2/3 phosphorylation in the fibroblast cells. Without NRP-1 (left panel), Smad1/5/8 is more phosphorylated and Smad2/3 less phosphorylated, and the corresponding gene expression controlled by the Smad proteins (e.g. Smad1/Id-1, Smad3/a-SMA, PAI-1) caused the cell to enter a less activated state (more quiescent). With NRP-1 (right panel), Smad1/5/8 is less phosphorylated, and Smad2/3 is more phosphorylated, and the corresponding gene expression controlled by the Smad proteins caused the cell to enter a more activated state (less quiescent).

Article Snippet: Antibodies and Other Reagents—NRP-1, Id-1, T RII, and -actin were from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA); Smad2, Smad5, p-Smad1/5, p-Smad2, p-Smad3 were purchased from Cell Signaling Technology, Inc. (Danvers, MA); -SMA was from Millipore (Billerica, MA); collagen I antibody was from Rockland Immunochemicals, Inc. (Gilbertsville, PA); PAI were fromNovus Bio. (Littleton, CO) TGF- 1 was from Biolegend (San Diego, CA); BMP9 was from R&D Systems (Minneapolis, MN); BMP2 and BMP4 were from Senway Biotech (San Diego,CA)ALK5 inhibitors SB431542 andALK5 inhibitors (2-(3- (6-methylpyridine-2-yl)-1H-pyrazol-4-yl)-1,5-naphthyridine) were from Stemgent (Cambridge, MA). siRNA and shRNA Transfection—siRNA for human NRP-1, T RII, and control were from Qiagen, Inc. (Valencia, CA). siRNA transfection was performed by Hiperfect (Qiagen) following the manufacturer’s instructions. shRNA for human NRP-1 and controls were from Open Biosystems (Huntsville, AL) and were prepared as previously described (18).

Techniques: Phospho-proteomics, Gene Expression

Journal: The Journal of Experimental Medicine

Article Title: Identification of a selective and direct NLRP3 inhibitor to treat inflammatory disorders

doi: 10.1084/jem.20171419

Figure Lengend Snippet: C172 inhibits NLRP3 activation via CFTR-independent manner. (A) C172 structure. (B) Immunoblot analysis of IL-1β and cleaved caspase-1 in culture supernatants (SN) of LPS-primed BMDMs treated with various doses (above lanes) of C172 and then stimulated with nigericin. (C and D) ELISA of IL-1β (C) and IL-18 (D) in supernatants from LPS-primed BMDMs treated with various doses (above lanes) of C172 and then stimulated with nigericin. (E) Immunoblot analysis of the indicated proteins in lysates from BMDMs treated with LPS for 3 h and stimulated with different doses of C172 for 30 min (C172 after LPS) or BMDMs treated with different doses of C172 for 30 min and then stimulated with LPS for 3 h (C172 before LPS). (F) ELISA of IL-1β in supernatants from LPS-primed WT or Cftr −/− BMDMs that were treated with nigericin with or without the presence of C172 (20 µM). Data are from three independent experiments with biological duplicates in each (C, D, and F; mean and SEM of n = 6) or are representative of three independent experiments (B and E). Statistics were analyzed using an unpaired Student’s t test: *, P < 0.05; **, P < 0.01; ***, P < 0.001.

Article Snippet: Recombinant human NLRP3 was from Novus Biologicals.

Techniques: Activation Assay, Western Blot, Enzyme-linked Immunosorbent Assay

Journal: The Journal of Experimental Medicine

Article Title: Identification of a selective and direct NLRP3 inhibitor to treat inflammatory disorders

doi: 10.1084/jem.20171419

Figure Lengend Snippet: CY-09 blocks NLRP3 inflammasome activation. (A) CY-09 structure. (B) Immunoblot analysis of IL-1β and cleaved caspase-1 (p20) in culture supernatants (SN) of LPS-primed BMDMs treated with various doses (above lanes) of CY-09 and then stimulated with nigericin and immunoblot analysis of the precursors of IL-1β (pro–IL-1β) and caspase-1 (pro–caspase-1) in lysates of those cells (input). (C–G) ELISA of IL-1β in supernatants from LPS-primed BMDMs treated with various doses of CY-09 and then stimulated with nigericin, MSU (C), ATP (D), cytosolic LPS (E), cytosolic poly(dA:dT) (F), or Salmonella (G). Data are from three independent experiments with biological duplicates in each (C–G; mean and SEM of n = 6) or are representative of three independent experiments (B). Statistics were analyzed using an unpaired Student’s t test: *, P < 0.05; ***, P < 0.001.

Article Snippet: Recombinant human NLRP3 was from Novus Biologicals.

Techniques: Activation Assay, Western Blot, Enzyme-linked Immunosorbent Assay

Journal: The Journal of Experimental Medicine

Article Title: Identification of a selective and direct NLRP3 inhibitor to treat inflammatory disorders

doi: 10.1084/jem.20171419

Figure Lengend Snippet: CY-09 inhibits NLRP3 inflammasome assembly. (A) Immunoblot analysis of ASC oligomerization in lysates of BMDMs treated with various doses (above lanes) of CY-09 and then stimulated with nigericin. SN, supernatants. (B) Confocal microscopy analysis in LPS-primed BMDMs treated with CY-09 (10 µM) and then stimulated with nigericin, followed by staining with MitoSOX and MitoTracker red. Nuclei were stained with DAPI. Bars, 10 µm. (C) Qualification of potassium efflux in LPS-primed BMDMs treated with different doses of CY-09 and then stimulated with nigericin. (D and E) Qualification of potassium (D) and chloride (E) efflux in LPS-primed BMDMs treated with different doses of indicated inhibitors and then stimulated with nigericin. (F) IP and immunoblot analysis of the interaction of Flag–NLRP3 and VSV–NLRP3 in the lysates of HEK-293T cells. (G) Immunoblot analysis of NLRP3 by SDD-AGE or SDS-PAGE assay in WT or Nlrp3 −/− BMDMs treated with various doses of CY-09 and then stimulated with nigericin. (H) IP and immunoblot analysis of the interaction of endogenous NLRP3 and ASC in LPS-primed BMDMs treated with various doses (above lanes) of CY-09 and then stimulated with nigericin. Data are from three independent experiments with biological duplicates in each (C–E; mean and SEM of n = 6) or are representative of three independent experiments (A, B, and F–H). Statistics were analyzed using an unpaired Student’s t test: *, P < 0.05; ***, P < 0.001.

Article Snippet: Recombinant human NLRP3 was from Novus Biologicals.

Techniques: Western Blot, Confocal Microscopy, Staining, SDS Page

Journal: The Journal of Experimental Medicine

Article Title: Identification of a selective and direct NLRP3 inhibitor to treat inflammatory disorders

doi: 10.1084/jem.20171419

Figure Lengend Snippet: CY-09 binds to the ATP-binding site of NLRP3 NACHT domain. (A) Cell lysates of LPS-primed BMDMs were incubated with different concentrations of biotin–CY-09, which were then pulled down with streptavidin beads. (B) Cell lysates of LPS-primed BMDMs or PMA-differentiated THP-1 cells were incubated with biotin–CY-09 and different concentrations of free CY-09, which were then pulled down with streptavidin beads. (C) Purified human NLRP3 protein was incubated with different concentrations of biotin–CY-09 and then pulled down with streptavidin beads. (D) Purified human NLRP3 protein was incubated with biotin–CY-09 and different concentrations of free CY-09, which were then pulled down with streptavidin beads. (E) MST assay for the affinity between CY-09 and purified GFP-NLRP3 protein. (F and G) Cell lysates from HEK-293T cells transfected with Flag-tagged NLRP3, NOD1, NOD2, AIM2, NLRC4, NLRP3–LRR, NLPR3–NACHT, or NLRP3–PYD constructs were incubated with indicated concentration of biotin–CY-09, which were then pulled down with streptavidin beads. Data are representative of three independent experiments.

Article Snippet: Recombinant human NLRP3 was from Novus Biologicals.

Techniques: Binding Assay, Incubation, Purification, Transfection, Construct, Concentration Assay

Journal: The Journal of Experimental Medicine

Article Title: Identification of a selective and direct NLRP3 inhibitor to treat inflammatory disorders

doi: 10.1084/jem.20171419

Figure Lengend Snippet: CY-09 inhibits NLRP3 ATPase activity. (A) ATPase activity assay for purified human NLRP3 in the presence of different concentrations of CY-09. (B) ATPase activity assay for purified Flag–NLRP3, NLRC4, NLRP1, NOD2 or RIG-I with or without presence of CY-09 (1 µM). (C) Cell lysates from HEK-293T cells transfected with Flag-tagged WT NLRP3 or NLRP3 constructs with Walker A or Walker B motif mutation were incubated with different concentrations of biotin–CY-09 and then pulled down with streptavidin beads. (D) ATP-binding assay for purified Flag–NLRP3 in the presence of different concentrations of CY-09. (E) ATPase activity assay for purified Flag–NLRP3 or mutants with or without presence of CY-09 (1 µM). (F) Docking complex of NLRP3 with CY-09. CY-09 is shown in sticks and colored green, while NLRP3 is shown in cartoon and colored light blue. Data are from three independent experiments with biological duplicates in each (A, B, and E; mean and SEM of n = 6) or are representative of two or three independent experiments (C and D). Statistics were analyzed using an unpaired Student’s t test: ***, P < 0.001.

Article Snippet: Recombinant human NLRP3 was from Novus Biologicals.

Techniques: Activity Assay, Purification, Transfection, Construct, Mutagenesis, Incubation, Binding Assay

Journal: The Journal of Experimental Medicine

Article Title: Identification of a selective and direct NLRP3 inhibitor to treat inflammatory disorders

doi: 10.1084/jem.20171419

Figure Lengend Snippet: CY-09 inhibits NLRP3 activation in MSU-induced peritonitis and a mouse model of MWS. (A and B) ELISA of IL-1β in the serum (A) or peritoneal cavity (B) of C57BL/6J mice intraperitoneally injected with MSU (1 mg/mouse) with or without CY-09 (40 mg/kg) or MCC950 (40 mg/kg). Data are representative of two independent experiments (mean and SEM of n = 6). (C) FACS analysis of neutrophil numbers in the peritoneal cavity of C57BL/6J mice intraperitoneally injected with MSU (1 mg/mouse) with or without CY-09 (40 mg/kg) or MCC950 (40 mg/kg). Data are representative of two independent experiments (mean and SEM of n = 6). (D) ELISA of IL-1β in serum of Nlrp3 −/− mice intraperitoneally injected with L. monocytogenes ( L.mon ; 5 × 10 6 ) for 8 h with or without CY-09 (40 mg/kg). (E) Weight of WT or Nlrp3A 350VneoR crossed with LysM-Cre mice ( NLRP3 -mut) treated with CY-09 or MCC950 at day 9. Data are representative of two independent experiments (mean and SEM). WT vehicle, WT CY-09, and WT MCC950 ( n = 6), NLRP3 -mut vehicle ( n = 4), NLRP3 -mut CY-09 or MCC950 ( n = 5). (F) NLRP3 -mut mice treated with CY-09 or MCC950 on day 9. (G) Survival of NLRP3 -mut mice treated with vehicle or CY-09 up to day 49 (CY-09 withdrawn at day 25). Data are representative of two independent experiments. CY-09 group ( n = 8), MCC950 group ( n = 8), vehicle group ( n = 9). Statistics were analyzed using an unpaired Student’s t test (A–E) or a generalized Wilcoxon test (G): ***, P < 0.001.

Article Snippet: Recombinant human NLRP3 was from Novus Biologicals.

Techniques: Activation Assay, Enzyme-linked Immunosorbent Assay, Injection

Journal: The Journal of Experimental Medicine

Article Title: Identification of a selective and direct NLRP3 inhibitor to treat inflammatory disorders

doi: 10.1084/jem.20171419

Figure Lengend Snippet: Treatment of metabolic disorders in HFD-induced diabetic mice with CY-09. (A and B) Food intake and body weight change of WT or Nlrp3 −/− mice that were first fed with an HFD for 14 wk and then treated with CY-09 for 6 wk. n = 6–8 per group. (C and D) Fasting (C) or fed (D) blood glucose concentrations at week 6 in the mice described in A. n = 6–8. (E) Fasting blood insulin concentration at week 6 in the mice described in A. n = 6–8 per group. (F–I) Glucose tolerance test (F and H) and insulin tolerance test (G and I) performed at week 6 in the mice described in A. n = 6–8 per group. (J) Representative H&E staining of liver sections of WT or Nlrp3 −/− mice that were first fed with an HFD for 14 wk and then treated with CY-09 for 6 wk. Bar, 50 µm; n = 6. Data are shown as mean and SEM and are representative of two independent experiments. Statistics were analyzed using an unpaired Student’s t test (A–E) or two-way ANOVA for (F–I): *, P < 0.05; **, P < 0.01; ***, P < 0.001.

Article Snippet: Recombinant human NLRP3 was from Novus Biologicals.

Techniques: Concentration Assay, Staining

Journal: The Journal of Experimental Medicine

Article Title: Identification of a selective and direct NLRP3 inhibitor to treat inflammatory disorders

doi: 10.1084/jem.20171419

Figure Lengend Snippet: CY-09 suppresses NLRP3-dependent metainflammation in diabetic mice. (A–H) WT or Nlrp3 −/− mice were first fed with an HFD for 14 wk and then treated with CY-09 for 6 wk. Plasma IL-1β (A) was assessed by ELISA. Liver (B, E, and G) and white adipose tissue (WAT; C, F, and H) were isolated and cultured for 24 h, and supernatants were analyzed by ELISA for IL-1β (B and C), TNF-α (E and F), or monocyte chemoattractant protein 1 (G and H). Caspase-1 activation in WAT was analyzed by immunoblot as indicated (D). n = 6–8 per group. Data are shown as mean and SEM and are representative of two independent experiments. Statistics were analyzed using an unpaired Student’s t test: *, P < 0.05; **, P < 0.01; ***, P < 0.001.

Article Snippet: Recombinant human NLRP3 was from Novus Biologicals.

Techniques: Clinical Proteomics, Enzyme-linked Immunosorbent Assay, Isolation, Cell Culture, Activation Assay, Western Blot

Journal: Nature communications

Article Title: A rationally designed JAZ subtype-selective agonist of jasmonate perception.

doi: 10.1038/s41467-018-06135-y

Figure Lengend Snippet: Fig. 1 One stereoisomer of COR is a potential JAZ subtype-selective agonist. a Chemical structures of jasmonate derivatives and coronatine diastereomers. b, c Schematic diagram of ligand-induced protein-protein interactions (PPIs) between COI1-JAZ co-receptors; b naturally occurring ligands (i.e., coronatine) can interact with all co-receptors, whereas c the stereochemical isomer (used in this study) can interact with only some co-receptors. d, e Pull down assay of purified GST-COI1 (5 nM) with recombinant proteins expressed in E. coli, including d MBP-JAZ1 (full length, approximately 40 nM), and e MBP-JAZ3 (full length, approximately 40 nM), in the presence of COR derivatives (100 nM). GST-COI1 bound to MBP-JAZ proteins was pulled down with amylose resin and analyzed by immunoblotting. Goat HRP-conjugated anti-GST antibody was used to detect GST-COI1 (black triangles). Rat anti-MBP antibody and goat HRP-conjugated rat-IgG antibody were used to visualize MBP-JAZ protein levels as the input materials (white triangles)

Article Snippet: The bound COI1-GST proteins was detected using anti-GST HRP conjugate (RPN1236, GE Healthcare, 2500-fold dilution in skimmed milk solution).

Techniques: Protein-Protein interactions, Pull Down Assay, Recombinant, Western Blot

Journal: Nature communications

Article Title: A rationally designed JAZ subtype-selective agonist of jasmonate perception.

doi: 10.1038/s41467-018-06135-y

Figure Lengend Snippet: Fig. 2 In vitro COI1-JAZs binding assay with epitope-conjugated JAZ short peptide. a Schematic of in vitro co-immunoprecipitation assay system for agonists of COI1-JAZ co-receptors using epitope-conjugated JAZ degron peptide and GST-COI1. An anti-fluorescein antibody was used to purify the ternary complex, and a GST-tag was used for the immunoblot assay with an anti-GST antibody. b Molecular design of the Oregon green (OG)-conjugated Jas motif of JAZ1. c Pull-down assay of purified GST-COI1 (5 nM) with each OG-conjugated JAZ peptide (10 nM) in the presence of stereoisomers of (+)-COR (3, ent3, 6, or ent6, 100 nM). d Pull-down assay of purified GST-COI1 (5 nM) with OG-conjugated JAZ13 peptide (10 nM) in the presence of 3, ent3, 6, or ent6 (100 nM). e Pull-down assay of purified GST-COI1 (5 nM) with OG-conjugated JAZ1 peptide (10 nM) in the presence of 3, ent3, 1 or 2 (100 nM); Goat HRP-conjugated anti-GST antibody was used to detect GST-COI1 in c–e

Article Snippet: The bound COI1-GST proteins was detected using anti-GST HRP conjugate (RPN1236, GE Healthcare, 2500-fold dilution in skimmed milk solution).

Techniques: In Vitro, Binding Assay, Co-Immunoprecipitation Assay, Western Blot, Pull Down Assay

Journal: Nature communications

Article Title: A rationally designed JAZ subtype-selective agonist of jasmonate perception.

doi: 10.1038/s41467-018-06135-y

Figure Lengend Snippet: Fig. 3 Rational design of JAZ subtype-selective agonist by an in silico docking study. a The obtained average structure of MD simulation of COI1-3-JAZ1. The hydrogen bond between the ketone group of 3 with A204JAZ1 or R496COI1 is indicated by a yellow dotted line. b The obtained average structure of COI1/JAZ9 complexed with ent6, which was constructed by in silico docking analyses and MD simulation. The hydrogen bond between the ketone group of ent6 with R496COI1 is shown as yellow dotted line. c The obtained average structure of COI1/JAZ10 complexed with ent6, which was constructed by in silico docking analyses and MD simulation. The hydrogen bond between the ketone group of ent6 with A170JAZ10 (corresponding to A204JAZ1) or R496COI1 is indicated by a yellow dotted line. d Synthesis scheme for compounds 7–9 from ent6 as a starting material and corresponding oxime molecules. e Pull-down assay of purified GST-COI1 (5 nM) with all OG-conjugated JAZ peptides (10 nM) in the presence of ent6, 7, 8, or 9 (500 nM). HRP-conjugated anti-GST antibody was used to detect GST-COI1. f Evaluation of GUS activity in the roots of 4-day-old 35S:JAZ1-GUS, 35S:JAZ9-GUS, and 35S:JAZ10-GUS plants. Seedlings were pretreated for 30 min with or without ligand (3, ent6, or 8, 1 µM), and stained with 5-bromo-4-chloro-3-indolyl glucuronide; the experiments were repeated three times with similar results. Scale bar, 1 mm. g Quantification of GUS activity in 20 roots of 4-d-old 35S:JAZ1-GUS, 35S: JAZ9-GUS, and 35S:JAZ10-GUS plants (n = 4). Significant differences were evaluated by one-way ANOVA/Tukey HSD post hoc test (p < 0.01). Seedlings were pretreated as described above. Three independent replicates were measured, and values represent mean ± s.d. (Supplementary Methods)

Article Snippet: The bound COI1-GST proteins was detected using anti-GST HRP conjugate (RPN1236, GE Healthcare, 2500-fold dilution in skimmed milk solution).

Techniques: In Silico, Construct, Pull Down Assay, Activity Assay, Staining