Journal: PloS one

Article Title: C-terminus of progranulin interacts with the beta-propeller region of sortilin to regulate progranulin trafficking.

doi: 10.1371/journal.pone.0021023

Figure Lengend Snippet: Figure 1. Progranulin interacts with the beta-propeller region of sortilin. (A) A schematic drawing of sortilin fragments cloned in the pDisplay vector (full length sortilin ecto domain (residues 81–757), sortilin 10CC region (residues 612–757) or sortilin beta propeller domain (residues 81–611)) and western blot to confirm their expression levels in COS-7 cells using anti-myc antibodies. (B) Binding of 100 nM alkaline phosphatase tagged progranulin (AP-PGRN) to COS-7 cells expressing various sortilin construct as indicated. Expression of these constructs was verified by anti- myc staining. (C) S283E mutation of sortilin abolished progranulin binding. Cell lysates from HEK293T cells transfected with vector control, wild type sortilin or the S283E mutant of sortilin were incubated with anti-Flag beads bound to Flag-PGRN. The presence of sortilin in the 3% input and anti- Flag IP was detected using anti-sortilin antidodies. (D) COS-7 cells expressing wild type sortilin or sortilin S283E mutant were allowed to bind to 100 nM AP-PGRN. The expression of S283E mutant was confirmed with western blot using anti-sortilin antibodies. Scale bar = 100 mm. doi:10.1371/journal.pone.0021023.g001

Article Snippet: S283E was generated using site directed mutagenesis using the sortilin cDNA from Origene as the template.

Techniques: Clone Assay, Plasmid Preparation, Western Blot, Expressing, Binding Assay, Construct, Staining, Mutagenesis, Transfection, Control, Incubation

Journal: PloS one

Article Title: C-terminus of progranulin interacts with the beta-propeller region of sortilin to regulate progranulin trafficking.

doi: 10.1371/journal.pone.0021023

Figure Lengend Snippet: Figure 2. Progranulin C-terminus mediates its binding to sortilin. (A) COS-7 cells expressing wild type sortilin were allowed to bind AP tagged progranulin fragments as indicated. (B) Quantification of the binding assay as described in (A). The intensity of AP staining was measured using the Metaexpress software (Y axis: average integrated optical density per cell (6100)). (C) C24 fragment of progranulin binds to sortilin with a similar affinity as the C100 fragment and full length progrnaulin. Sortilin expressing COS-7 cells were allowed to bind to AP tagged C100 and C24 at

Article Snippet: S283E was generated using site directed mutagenesis using the sortilin cDNA from Origene as the template.

Techniques: Binding Assay, Expressing, Staining, Software

Journal: PloS one

Article Title: C-terminus of progranulin interacts with the beta-propeller region of sortilin to regulate progranulin trafficking.

doi: 10.1371/journal.pone.0021023

Figure Lengend Snippet: Figure 3. Progranulin C-terminal peptide displaces progranulin binding to sortilin. (A) Alkaline phosphatase fused full length PGRN and C24 binding to sortilin can be displaced by purified recombinant his-PGRN, C18 peptide (EAPRWDAPLRDPALRQLL) but not by his- PGRND3aa. Sortilin expressing COS-7 cells were incubated with 150 nM purified his-PGRN, his-PGRND3aa or C18 peptide of indicated concentrations for 1 hour before adding 10 nM of AP-PGRN or AP-C24. (B) Quantification of binding intensity for experiments in (A) using the ImageXpress system. Scale bar = 100 mm. *, p,0.05;**, p,0.01, n = 3–6, paired Student’s T-test. doi:10.1371/journal.pone.0021023.g003

Article Snippet: S283E was generated using site directed mutagenesis using the sortilin cDNA from Origene as the template.

Techniques: Binding Assay, Purification, Recombinant, Expressing, Incubation

Journal: PloS one

Article Title: C-terminus of progranulin interacts with the beta-propeller region of sortilin to regulate progranulin trafficking.

doi: 10.1371/journal.pone.0021023

Figure Lengend Snippet: Figure 4. The C-terminal carboxylate of progranulin mediates its binding to sortilin. (A) Illustration of binding of progranulin C- terminal tail to sortilin. Drawing was made with Pymol software. Residue backbones of sortilin are labeled in gray and progranulin labeled in green. (B) Alignment of the C-terminal residues of progranulin from different species of mammals (from top to bottom: gi|4504151|[Homo sapiens], gi|114666849|[Pan troglodytes], gi|296476263|[Bos taurus], gi|224967126|[Mus musculus], gi|204224|[R- attus norvegicus] ) and neurotensin. (C) Mouse progranulin interacts with mouse sortilin through its C-terminal tail. COS-7 cells transfected with mouse sortilin were incubated with 10 or 50 nM of AP tagged C24 and C21 of mouse progranulin (mC24 and mC21) or C24 of human progranulin (hC24). Scale bar = 100 mm. doi:10.1371/journal.pone.0021023.g004

Article Snippet: S283E was generated using site directed mutagenesis using the sortilin cDNA from Origene as the template.

Techniques: Binding Assay, Software, Residue, Labeling, Transfection, Incubation

Journal: PloS one

Article Title: C-terminus of progranulin interacts with the beta-propeller region of sortilin to regulate progranulin trafficking.

doi: 10.1371/journal.pone.0021023

Figure Lengend Snippet: Figure 5. Deletion of progranulin C-terminal tail abolishes sortilin dependent control of progranulin trafficking but does not affect progranulin secretion. (A) Deletion of the PGRN C-terminal 3 residues (PGRND3aa) or S283E mutation in sortilin abolishes sortilin dependent control of PGRN trafficking. AP tagged full length progranulin (FL), PGRND3aa (-3aa) or AP alone were co-transfected with pcDNA vector control, wild type or S283E mutant of sortilin into HEK 293T cells or N2A cells. The AP activities in the medium were measured 4 days after transfection. The AP activities in sortilin expressing cells were expressed as percentage of vector control. *, p,0.05;**, p,0.01, n = 3–5, paired Student’s T-test. (B) Deletion of progranulin C-terminal residues does not affect progranulin secretion. Progranulin expression constructs as AP fusion proteins were transfected in HEK293T and N2Acells. Progranulin levels in the cell lysates and media were determined by western blots using anti-AP antibodies. doi:10.1371/journal.pone.0021023.g005

Article Snippet: S283E was generated using site directed mutagenesis using the sortilin cDNA from Origene as the template.

Techniques: Control, Mutagenesis, Transfection, Plasmid Preparation, Expressing, Construct, Western Blot

Journal: Biology

Article Title: ProBDNF Upregulation in Murine Hind Limb Ischemia Reperfusion Injury: A Driver of Inflammation.

doi: 10.3390/biology12070903

Figure Lengend Snippet: Figure 2. Comparison of protein levels of BDNF, proBDNF, and their receptors in skeletal muscle and brain. (a) Representative Western blot images comparing protein levels of proBDNF and ma- ture BDNF in fast twitch (EDL) and slow twitch (soleus) skeletal muscle compared to the brain. (b) Quantification of Western blot densitometry of proBDNF and mature BDNF as a ratio to total protein loading control compared using one-way ANOVA followed by Tukey’s post hoc test n = 4 per group, * p < 0.05. (c) Representative Western blot images comparing protein levels of receptors TrkB, p75NTR, and sortilin in fast twitch (EDL) and slow twitch (soleus) skeletal muscle compared to the brain. (d) Quantification of Western blot densitometry of TrkB, p75NTR, and sortilin as a ratio to total protein loaded and compared using one-way ANOVA followed by Tukey’s post hoc test n = 4 per group, * p < 0.05, ** p < 0.01, **** p < 0.0001.

Article Snippet: Primary antibodies used were p75NTR (1:1000 Cell Signaling Technologies, 8238S), TrkB (1:1000, ProteinTech, 13129- 1-AP), sortilin (1:1000, ProteinTech, 12369-1-AP), proBDNF (1:500 Santa Cruz, sc-6551), BDNF (1:1000 Abcam, ab108319), Furin (1:1000 ABclonal, A7445), tPA (1:1000 ABclonal, A4210), MCP-1 (1:1000 Cell Signaling Technology, 2029S), IFN-γ (Cell Signaling Technology, 3159S), TNFα (1:500 Santa Cruz, sc-8301), and TGF-β (1:1000 Biolegend, 141402).

Techniques: Comparison, Western Blot, Control

Journal: International Journal of Molecular Sciences

Article Title: Neurotrophins and Trk Neurotrophin Receptors in the Retina of Adult Killifish ( Nothobranchius guentheri )

doi: 10.3390/ijms25052732

Figure Lengend Snippet: Details of antibodies.

Article Snippet: NT-3 (A4) , Santa Cruz Biotechnology, Inc., Dallas, TX, USA , sc-518099 , mouse , 1:100 , .

Techniques: