fz8  (Thermo Fisher)

Journal: International Journal of Molecular Sciences

Article Title: Açai Berry Administration Promotes Wound Healing through Wnt/β-Catenin Pathway

doi: 10.3390/ijms24010834

Figure Lengend Snippet: Western blot analysis of Wnt3a ( A ), FZ8 ( B ), cytosolic β-catenin ( C ), and nuclear β-catenin ( D ). A demonstrative blot of lysates with a densitometric analysis for all animals is shown. Data are expressed as the mean ± SEM of N = 6 mice/group. ** p < 0.01 vs. sham, *** p < 0.001 vs. sham; # p < 0.05 vs. WH; ## p < 0.01 vs. WH.

Article Snippet: Western blot analysis was realized as previously described [ ]; anti-IκB-α (1:1000, SCB, #sc1643), anti-NF-κB p65 (1:1000; SCB, #sc8414) anti-Wnt3a (Santa Cruz Biotechnology (SCB), Dallas, TX, USA, sc-80457), anti-FZ8 (Bioworld Technology, St. Louis Park, MN, USA).

Techniques: Western Blot

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: Frizzled 1 and Wnt1 as new potential therapeutic targets in the traumatically injured spinal cord

doi: 10.1007/s00018-019-03427-4

Figure Lengend Snippet: Primary and secondary antibodies used for immunohistochemistry

Article Snippet: Table 2 Tissue species Immunohistochemical method Antibody Reference Dilution Rat Chromogen-based simple immunohistochemistry Rb anti-Fz1 ab71342, Abcam 1:250 Rb anti-Iba1 019-19741, Wako 1:1000 Mo anti-GFAP G3893, Sigma-Aldrich 1:1000 Rb anti-NG2 AB5320, Millipore 1:250 Biotinilated Go anti-Rb BA-1000, Vector 1:500 Biotinilated Ho anti-Mo BA-2001, Vector 1:500 Fluorescence-based simple immunohistochemistry Mo anti-APC OP80, Calbiochem 1:100 Rb anti-NeuN MABN140, Millipore 1:100 Rb anti-serotonin S5545, Sigma-Aldrich 1:500 Dylight 594-linked Go anti-Rb ab96897, Abcam 1:500 Dylight 594-linked Go anti-Mo ab96881, Abcam 1:500 Fluorescence-based double immunohistochemistry Rb anti-Fz1 ab71342, Abcam 1:50 Mo anti-GFAP G3893, Sigma-Aldrich 1:1000 Mo anti-NeuN MABN140, Millipore 1:100 Mo anti-APC OP80, Calbiochem 1:100 Mo anti-NF200 N0142, Sigma-Aldrich 1:2000 Mo anti-OX42 ab58457, Abcam 1:500 Mo anti-NG2 37-2700, Zymed 1:100 Mo anti-RECA1 MCA970GA, AbD Serotec 1:250 Dylight 594-linked Go anti-Rb ab96897, Abcam 1:500 Dylight 594-linked Go anti-Mo ab96881, Abcam 1:500 Human Chromogen-based simple immunohistochemistry Rb anti-Fz1 ab71342, Abcam 1:500 Rb anti-Fz2 ab94913, Abcam 1:500 Rb anti-Fz3 ab188974, Abcam 1:50 Rb anti-Fz4 ab83042, Abcam 1:1000 Rb anti-Fz5 ab75234, Abcam 1:500 Rb anti-Fz6 ab150545, Abcam 1:100 Rb anti-Fz7 ab64636, Abcam 1:1000 Rb anti-Fz8 ab75235, Abcam 1:50 Rb anti-Fz9 ab61430, Abcam 1:50 Rb anti-Fz10 ab83044, Abcam 1:500 Rb anti-Ryk AP7677a, Abgent 1:500 Rb anti-Ror1 AP7671d, Abgent 1:50 Rb anti-Ror2 AP7672d, Abgent 1:50 Mo anti-PTK7 400005754-M06, Abnova 1:500 Rb anti-pLRP6 (Ser 1490) 2568L, Cell Signaling 1:100 Biotinilated Go anti-Rb BA-1000, Vector 1:500 Biotinilated Ho anti-Mo BA-2001, Vector 1:500 Fluorescence-based double immunohistochemistry Rabbit anti-Fz1 ab71342, Abcam 1:100 Mo anti-GFAP G3893, Sigma-Aldrich 1:1000 Mo anti-CNPase SMI-91R, Covance 1:100 Mo anti-CD31 #3528, Cell Signaling 1:100 Mo Pan Neuronal Marker MAB2300, Millipore 1:50 Go anti-Iba1 ab5076, Abcam 1:100 Dylight 594-linked Go anti-Rb DI-1594, Vector 1:500 Dylight 488-linked Ho anti-Mo DI-2488, Vector 1:500 Alexa 488-linked Do anti-Go A11055, Invitrogen 1:1000 Open in a separate window Primary and secondary antibodies used for immunohistochemistry. .

Techniques: Immunohistochemical staining, Immunohistochemistry, Plasmid Preparation, Fluorescence, Marker

Journal: Nature structural & molecular biology

Article Title: Receptor subtype discrimination using extensive shape complementary designed interfaces

doi: 10.1038/s41594-019-0224-z

Figure Lengend Snippet: DRPB_Fz8-Fz8CRD complex structure confirms computational design and paves the way for specificity tuning. a, Crystal structure of DRPB_Fz8-Fz8CRD matches the computationally designed complex. The Fz8CRD is shown by blue surface. The crystal complex DRPB_Fz8 is colored in light orange and the computational model is colored in grey. The Ala “fingers” of DRPB_Fz8 are deeply “grasping” the hydrophobic groove of Fz8CRD. b, Overall structures of DRPB_Fz8 with Fz8CRD. c, An “open-book” view showing the interacting surface on both Fz8CRD and DRPB_Fz8. The interacting surface on Fz8CRD and DRPB_Fz8 is colored in grey. d, The “palm” region of DRPB_Fz8 consists of hydrophobic residues that make van der Waals interactions with Fz8 CRD. The “fingers” region of DRPB_Fz8 is omitted to gain a clear view. e, The “wrist” region of DRPB_Fz8 consists of hydrophilic and charged residues that contribute to both solubility and receptor recognition. The DRPB_Fz8 Asn112 and Arg145 form hydrogen bonds and salt bridges with Glu76 of Fz8CRD, shown as grey dashed lines.

Article Snippet: The CRDs of human FZ1 (residues 113–182), FZ4 (residues 42–161), FZ7 (residues 36–163) and FZ8 (residues 32–150 with N49Q mutation) used for crystallization contained a C-terminal 6X His tag were expressed in High Five ( Trichoplusia ni ) cells (Invitrogen) using the baculovirus expression system.

Techniques: Solubility

Journal: Nature structural & molecular biology

Article Title: Receptor subtype discrimination using extensive shape complementary designed interfaces

doi: 10.1038/s41594-019-0224-z

Figure Lengend Snippet: Complex structures of DRPB-Fz explain Fz subtype specificity. a, Titration of DPRB_Fz7/8 to biotinylated Fz 1/2/4/5/7/8 CRDs. DPRB_Fz8 was expressed on yeast surface. Biotinylated Fz 1/2/4/5/7/8 was added at gradient concentration. Alexa Fluor® 647 streptavidin was subsequently added and the median fluorescence intensity (MFI) was analyzed and plotted in Prism 7. DPRB_Fz7/8 showed EC 50 of 19.0, 31.6 and 5.6 nM to Fz1, 2 and 7. DPRB_Fz7/8 showed weaker staining to Fz5/8 with EC 50 not available. Representative Ala111Asp mutation from DPRB_Fz8 to DPRB_Fz7/8 allows hydrogen bond and salt bridge formation between DPRB_Fz7/8 with Fz7 Lys91, shown by grey dashed lines. DPRB_Fz7/8 is colored in pale cyan. Fz7CRD is colored in pink and Fz8CRD is colored in slate. The corresponding position of Lys91 is Glu, weakening DPRB_Fz7/8 interaction with Fz8 subtype. b, DPRB_Fz7 showed strong binding to Fz1, 2 and 7, respectively. DPRB_Fz7 showed no cross-reactivity to Fz5/8 up to 1 uM concentration ( , ). The Ala108Asp mutation from DPRB_Fz7/8 to DPRB_Fz7 leads to steric clashes with Trp73 of Fz8 CRD. On the other hand, the corresponding position of Fz7 subtype is Tyr, whose less bulky sidechain allows it take a different rotamer conformation. This Ala108Asp mutation thus further confers specificity to Fz7 subtype by eliminating Fz8 binding. DPRB_Fz7/8 here is colored in light cyan while DPRB_Fz7 is colored in cyan. c, DPRB_Fz4 only binds to Fz4 with EC 50 of 1.6 nM. DPRB_Fz4-Fz4CRD showed backbone movement compared with DPRB_Fz8-Fz8CRD. Therefore, DPRB_Fz4 and Fz4CRD were individually superpositioned with DPRB_Fz8 and Fz8CRD. The differences between DPRB_Fz4 and DPRB_Fz8 surface lead to less favorable interactions between DPRB_Fz4 with Fz8. For examples, Asp133Gly, Phe141Ile and Trp144Phe weaken interactions to Fz8CRD due to less bulky sidechains. The yeast titration experiements were repeated once with similar results.

Article Snippet: The CRDs of human FZ1 (residues 113–182), FZ4 (residues 42–161), FZ7 (residues 36–163) and FZ8 (residues 32–150 with N49Q mutation) used for crystallization contained a C-terminal 6X His tag were expressed in High Five ( Trichoplusia ni ) cells (Invitrogen) using the baculovirus expression system.

Techniques: Titration, Concentration Assay, Fluorescence, Staining, Mutagenesis, Binding Assay

Journal: Nature structural & molecular biology

Article Title: Receptor subtype discrimination using extensive shape complementary designed interfaces

doi: 10.1038/s41594-019-0224-z

Figure Lengend Snippet: Data collection and refinement statistics (molecular replacement)

Article Snippet: The CRDs of human FZ1 (residues 113–182), FZ4 (residues 42–161), FZ7 (residues 36–163) and FZ8 (residues 32–150 with N49Q mutation) used for crystallization contained a C-terminal 6X His tag were expressed in High Five ( Trichoplusia ni ) cells (Invitrogen) using the baculovirus expression system.

Techniques:

Journal: Nature structural & molecular biology

Article Title: Receptor subtype discrimination using extensive shape complementary designed interfaces

doi: 10.1038/s41594-019-0224-z

Figure Lengend Snippet: Fz-subtype specific DRPB antagonists inhibit homeostasis of the intestinal crypt stem cell compartment and expression of liver Wnt target genes. a, Mouse (top) and human (bottom) primary duodenal organoids which endogenously produce Wnt were cultured in submerged Matrigel in medium containing ENR (EGF/Noggin/R-spondin) with different Fz-subtype-specific DRPB antagonist at 100 nM. The images were taken at day 7 (mouse) or day 10 (human) of continuous DRPB antagonist exposure. Each Fz antagonist was replenished every 3 days during medium change. In vitro experiments were repeated at least three times, and representative images are shown. b, In vivo phenotype following daily intravenous (i.v.) injections of recombinant MSA-DRPB_Fz8 at a concentration of 20mg/kg or PBS for 7 days. H & E staining of jejunum is shown. The experiment was performed once due to limited protein yield. c-d, Representative images of H & E staining of jejunum cross-sections ( c) and glutamine synthetase (GLUL, pericentral marker) immunofluorescence staining of livers ( d) from mice that received adenoviruses expressing negative control IgG2a Fc (Fc) or different Fz-subtype-specific DRPB antagonist, 7 days post adenovirus injection (representative images from n=6 to n=9 mice/group. Experiments were repeated twice). e, Quantification of the GLUL immunofluorescence signal intensity shown in (D). f-g, Quantification of the Glul (f) and Axin2 (g) transcript levels relative to GAPDH from liver samples ( d ) by qRT-PCR. Statistical analysis was performed using GraphPad Prism software. Data represent mean ± s.e.m. (n=9; control and n=6; other groups biological replicates from 2 experiments; for all in vivo experiments (panels c-g)) Two-sided P-values were calculated by Dunn’s test of multiple comparisons following Kruskal-Wallis test. *P < 0.05; **P < 0.01; ****P < 0.0001.

Article Snippet: The CRDs of human FZ1 (residues 113–182), FZ4 (residues 42–161), FZ7 (residues 36–163) and FZ8 (residues 32–150 with N49Q mutation) used for crystallization contained a C-terminal 6X His tag were expressed in High Five ( Trichoplusia ni ) cells (Invitrogen) using the baculovirus expression system.

Techniques: Expressing, Cell Culture, In Vitro, In Vivo, Recombinant, Concentration Assay, Staining, Marker, Immunofluorescence, Negative Control, Injection, Quantitative RT-PCR, Software, Control

Journal: Developmental cell

Article Title: APC Inhibits Ligand-Independent Wnt Signaling by the Clathrin Endocytic Pathway

doi: 10.1016/j.devcel.2018.02.013

Figure Lengend Snippet: (A–D) Fz8-Fc blocks Wnt ligand-mediated signaling but not signaling in APC-deficient cells. (A) RKO STF cells were incubated with Wnt3a and Fz8-Fc at the indicated concentrations. (B) HEK293 STF cells transfected with APC siRNA and (C) RKO APCKO cells were incubated with Fz8-Fc. (D) Wnt activation in CRC cells is not dependent on Wnt ligand. CRC cells were incubated for 48 hr with Fz8-Fc at the indicated concentrations.

Article Snippet: For inhibitor studies, cells were incubated with mAb7E5, Wnt-C59 (Cellagen Technology), IWP-2 (StemRD) or Fz8-Fc (R&D Systems) for 24 h (or 48 h for SW480, DLD1, and HCT116 cells) prior to lysis.

Techniques: Incubation, Transfection, Activation Assay

Journal: Developmental cell

Article Title: APC Inhibits Ligand-Independent Wnt Signaling by the Clathrin Endocytic Pathway

doi: 10.1016/j.devcel.2018.02.013

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: For inhibitor studies, cells were incubated with mAb7E5, Wnt-C59 (Cellagen Technology), IWP-2 (StemRD) or Fz8-Fc (R&D Systems) for 24 h (or 48 h for SW480, DLD1, and HCT116 cells) prior to lysis.

Techniques: Control, Virus, Recombinant, Luciferase, Viability Assay, Reverse Transcription, Plasmid Preparation, Software