Journal: Structure (London, England : 1993)

Article Title: Functional Relevance of IL-1 Receptor Inter-domain Flexibility for Cytokine Binding and Signaling

doi: 10.1016/j.str.2019.05.011

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Rabbit Anti-IL-1RAcP Antibody , Rockland , Cat# 600–401-BV3; RRID:AB_2613201.

Techniques: Recombinant, Plasmid Preparation, Software

Journal: Scientific Reports

Article Title: Somatostatin receptor expression on von Hippel-Lindau-associated hemangioblastomas offers novel therapeutic target

doi: 10.1038/srep40822

Figure Lengend Snippet: Patients with von Hippel Lindau disease (VHL) undergoing surgical resection of posterior fossa hemangioblastoma provide confirmation of DOTATATE avidity of these lesions. The post-operative T1 weighted MRI image ( A ) and DOTATATE PET image ( B ) confirm complete resection of a DOTATATE avid posterior fossa hemangioblastoma ( C , D ). Representative images ( E ) of anti-SSTR IHC staining classified as negative (i), positive/negative (ii), positive (iii), double positive (iv) or triple positive (v). Scale bar = 50 μm. Average staining intensity for each SSTR subtype is shown in panel F. Number of tumors with SSTR expression are categorized by receptor subtype ( G ). Positive staining patterns were found in 8/9 tumors against SSTR1, in 8/9 tumors against SSTR2a, in 2/9 tumors against SSTR3, in 9/9 tumors against SSTR4, and in 8/9 tumors in SSTR5.

Article Snippet: Antibodies used were anti-Somatostatin Receptor 1 (SSTR1) (1:125, LS-C332380, Lifespan Bio, Seattle, WA), anti-Somatostatin Receptor 2a (SSTR2a) (1:500, PA3-109, Thermo Fischer Scientific Inc., Rockford, IL), anti-Somatostatin Receptor 3 (SSTR3) (1:100, TA323276, Origene, Rockville, MD), anti-Somatostatin Receptor 4 (SSTR4) (1:500, GTX70677, Genetex, Irvine, CA) anti-Somatostatin Receptor 5 (1:50, ab109495, Abcam, Cambridge, MA).

Techniques: Immunohistochemistry, Staining, Expressing

Journal: Scientific Reports

Article Title: Somatostatin receptor expression on von Hippel-Lindau-associated hemangioblastomas offers novel therapeutic target

doi: 10.1038/srep40822

Figure Lengend Snippet: A total of 9 VHL-HBs were stained against somatostatin receptor subtypes 1, 2a, 3, 4, and 5.

Article Snippet: Antibodies used were anti-Somatostatin Receptor 1 (SSTR1) (1:125, LS-C332380, Lifespan Bio, Seattle, WA), anti-Somatostatin Receptor 2a (SSTR2a) (1:500, PA3-109, Thermo Fischer Scientific Inc., Rockford, IL), anti-Somatostatin Receptor 3 (SSTR3) (1:100, TA323276, Origene, Rockville, MD), anti-Somatostatin Receptor 4 (SSTR4) (1:500, GTX70677, Genetex, Irvine, CA) anti-Somatostatin Receptor 5 (1:50, ab109495, Abcam, Cambridge, MA).

Techniques: Staining

Journal: Development (Cambridge, England)

Article Title: Ccbe1 regulates Vegfc-mediated induction of Vegfr3 signaling during embryonic lymphangiogenesis.

doi: 10.1242/dev.100495

Figure Lengend Snippet: Fig. 2. ccbe1, vegfc and vegfr3 genetically interact in double and triple heterozygous animals. (A-H) Confocal projections of Tg(fli1a:EGFP; kdr- l:mCherry) show grossly unaltered overall morphology and blood vasculature in ccbe1hu3613 (B), vegfr3hu4602 (E) and vegfchu5055 (F) mutants compared with wild type (A). The TD (C, arrows) is absent in ccbe1hu3613 (D), vegfr3 hu4602 (G) and vegfc hu5055 (H) mutants (asterisks). (I-K) ccbe1, vegfc and vegfr3 genetically interact in double heterozygote embryos, which display lymphatic defects. Offspring from vegfc+/−and vegfr3+/−carriers give rise to 28% of embryos (n=28/99) with a TD length of ≤50%. This population is significantly enriched (71%; n=20/28; P<0.0001) in double heterozygotes (I). Similarly in ccbe1+/−and vegfr3+/−

Article Snippet: Standard western blotting approaches were used to detect Vegfr3 with both anti-phospho-VEGFR3 (1/1000; Cell Applications, CB5793) and anti (non-phosphorylated) VEGFR3 (1/1000; Cell Applications, CB5792).

Techniques:

Journal: Development (Cambridge, England)

Article Title: Ccbe1 regulates Vegfc-mediated induction of Vegfr3 signaling during embryonic lymphangiogenesis.

doi: 10.1242/dev.100495

Figure Lengend Snippet: Fig. 3. Phenotypes driven by ectopic Vegfc/Vegfr3 signaling are suppressed in ccbe1-deficient embryos. (A) At 72 hpf, dll4 morphants display an arterial hyperbranching phenotype (arrow) driven by increased Vegfc/Vegfr3 signaling in the transgenic Tg(fli1a:EGFP) line. This phenotype was suppressed in ccbe1hu3613 mutants. Eighty-one per cent of MO-dll4 injected embryos displaying wild-type or mild phenotypes were ccbe1 mutants (n=17/21), whereas the population displaying the most severe phenotype was mainly composed of wild-type or heterozygous siblings (83%; n=139/168). (B) In dll4 morphants, arteries are sensitized to increased vegfc expression during primary sprouting. Arteries in MO-dll4, vegfc mRNA-injected embryos display aberrant, ectopic turning (arrow) as early as 30 hpf. Embryos from ccbe1 carrier incrosses, injected with 100 ng vegfc mRNA and 5 ng MO-dll4, were sorted into the phenotypic categories ‘wild type’ and ‘severe’. Genotyping revealed that 70% of the embryos displaying wild-type morphology were ccbe1 mutants (n=19/27). By contrast, the population affected by the most severe phenotype was composed of 83% wild-type or heterozygous siblings (n=122/147). (C) Confocal projections of Tg(fli1a:EGFP; flt1:tomato; hsp70l:Gal;4XUAS:vegfc) embryos show that endothelial cells in heat-shocked embryos display aberrant ectopic branching at 72 hpf. The ectopic endothelial cells are venous derived (flt1:tomato negative, arrow in Ciii). Heat-shocked embryos that were injected with 2.5 ng of MO-ccbe1 do not show this phenotype (asterisks). Scoring of the number of aberrant vISVs per heat-shocked embryo showed a significant rescue (0.12 in MO-ccbe1 injected n=22, versus 4.76 in uninjected controls n=25; P<0.0001) of the phenotype.

Article Snippet: Standard western blotting approaches were used to detect Vegfr3 with both anti-phospho-VEGFR3 (1/1000; Cell Applications, CB5793) and anti (non-phosphorylated) VEGFR3 (1/1000; Cell Applications, CB5792).

Techniques: Transgenic Assay, Injection, Expressing, Derivative Assay

Journal: Development (Cambridge, England)

Article Title: Ccbe1 regulates Vegfc-mediated induction of Vegfr3 signaling during embryonic lymphangiogenesis.

doi: 10.1242/dev.100495

Figure Lengend Snippet: Fig. 4. Vegfr3-dependent Erk signaling requires ccbe1 during the induction of secondary sprouting in zebrafish. (A) Analysis of phospho-Erk (P-Erk) expression in 32 hpf embryos. P-Erk (green) and fli1a:EGFP (white) images (lateral view) show P-Erk detected broadly in whole-mount and cross-sectioned (right-hand panels, merge upper, P-Erk lower) control embryos. Signal was increased in Vegfc-induced (dll4 MO + vegfc mRNA-injected) embryos in the posterior cardinal vein (n=8/8; Vegfc-induced embryos all showed ectopic expression in the ventral wall of the PCV). Cross section merged channel images shown in a and b, P-Erk only in c and d. Treatment with the Erk inhibitor PD98059 led to a reduction in all P-Erk staining. Arrows indicate P-Erk expression in the dorsal PCV. DA (dorsal aorta) and PCV (posterior cardinal vein) are indicated. (B) Comparison of P-Erk staining in control uninjected (left), MO-vegfr3 and MO-ccbe1 embryos. Upper panels are merged images and lower P-Erk only, viewed laterally (left) and cross-sectioned (right). Cross sections (right) are from separate embryos. Arrows indicate P-Erk expression in the dorsal PCV. DA and PCV are indicated. (C) Quantification of P-Erk-positive cells in the cardinal vein located in the dorsal compared with ventral wall (left-hand graph). Scores through individual sections of z-stack images from 12 control embryos, scored laterally across three somites in the trunk. Quantification of P-Erk-positive cells in the cardinal vein in control and MO-injected conditions (right-hand graph) (scores from n=10 control embryos, n=13 MO-vegfr3-injected and n=15 MO-ccbe1-injected embryos). (D) Immunoprecipitation (IP) and western blot (IB) detection of phosphorylated Vegfr3 at 32 hpf in wild type and in ccbe1, vegfr3, vegfc morphant and vegfc mRNA-injected embryos. The level of phosphorylated Vegfr3 is markedly reduced in ccbe1, vegfr3 and vegfc morphants, but is increased in vegfc-mRNA injected (500 ng) embryos compared with wild type (D, upper blot, IP for phospho-Vegfr3 and IB detection with phospho-Vegfr3). Loading controls were: the IgG light chain [IgG(l)] present in all blots after IP (D, middle blot), and Myosin to monitor protein input in IPs (D, lower blot). Quantification of Vegfr3 phosphorylation (relative to the loading control) based on three independent experiments is shown in right-hand panel. The decrease in MO-ccbe1 compared with uninjected controls is statistically significant (P<0.05). (E) qPCR analysis of the expression of ccbe1, vegfr3, vegfc, kdr and kdrl in uninjected control and MO-ccbe1-, MO-vegfc-, and MO-vegfr3-injected embryos. Error bars represent s.d. (C) or s.e.m. (D,E).

Article Snippet: Standard western blotting approaches were used to detect Vegfr3 with both anti-phospho-VEGFR3 (1/1000; Cell Applications, CB5793) and anti (non-phosphorylated) VEGFR3 (1/1000; Cell Applications, CB5792).

Techniques: Expressing, Control, Injection, Staining, Comparison, Immunoprecipitation, Western Blot, Phospho-proteomics

Journal: Development (Cambridge, England)

Article Title: Ccbe1 regulates Vegfc-mediated induction of Vegfr3 signaling during embryonic lymphangiogenesis.

doi: 10.1242/dev.100495

Figure Lengend Snippet: Fig. 6. Ectopic expression of mature VEGFC rescues secondary sprouting in ccbe1 morphants. (A) Confocal projections of Tg(fli1a:EGFP) at 54 hpf. Knock down of ccbe1 or vegfr3 leads to a loss of PLs at the horizontal myoseptum (arrowheads and asterisks). Ectopic expression of the mature form of VEGFC strongly rescues PL formation in ccbe1 morphants but not in vegfr3 morphants. Arrows indicate hyperbranched ISVs. (B) Quantification of PL formation at 54 hpf. In wild type, 98% (n=54/55) of embryos develop PLs, whereas in MO- ccbe1-injected embryos <4% (n=2/52) do. PL development is rescued to 74% (n=29/39) in ccbe1 morphants transiently overexpressing ΔNΔCVEGFC (P<0.0001). This rescue was never observed in vegfr3 morphants with all embryos devoid of PLs (n=23/23). (C) Quantification of ISV hypersprouting at 54 hpf. ISV hypersprouting was observed in wild-type embryos (93%; n=40/43), with mild reductions in ccbe1 morphants (79%; n=31/39) and vegfr3 morphants (65%; n=15/23) after ΔNΔCVEGFC overexpression.

Article Snippet: Standard western blotting approaches were used to detect Vegfr3 with both anti-phospho-VEGFR3 (1/1000; Cell Applications, CB5793) and anti (non-phosphorylated) VEGFR3 (1/1000; Cell Applications, CB5792).

Techniques: Expressing, Knockdown, Injection, Over Expression

Journal: Development (Cambridge, England)

Article Title: Ccbe1 regulates Vegfc-mediated induction of Vegfr3 signaling during embryonic lymphangiogenesis.

doi: 10.1242/dev.100495

Figure Lengend Snippet: Fig. 7. Ccbe1 activates Vegfc to induce Vegfr3 signaling. Proposed model for coordination of angiogenesis by Ccbe1, Vegfc and Vegfr3 in the developing embryo. Vegfc is produced in a largely inactive full-length form that is processed and released from the cell surface/ECM in a Ccbe1- dependent manner to generate the mature, highly active form. Downstream, arteries respond in a manner dampened by Dll4-dependent suppression of Vegfr3 signaling (Hogan et al., 2009b), whereas Vegfr3 signaling in veins induces secondary angiogenesis, which produces both intersegmental veins and lymphatic vascular precursor cells.

Article Snippet: Standard western blotting approaches were used to detect Vegfr3 with both anti-phospho-VEGFR3 (1/1000; Cell Applications, CB5793) and anti (non-phosphorylated) VEGFR3 (1/1000; Cell Applications, CB5792).

Techniques: Produced