Journal: mAbs

Article Title: Assessing kinetic and epitopic diversity across orthogonal monoclonal antibody generation platforms

doi: 10.1080/19420862.2015.1118596

Figure Lengend Snippet: Affinity comparison of mAbs derived from chicken immunizations and other sources for 2 unrelated model antigens, PCSK9 and PGRN. (A) Biacore binding curves and global fits of select anti-PCSK9 mAbs from chicken immunization showing a diverse set of kinetic profiles. The colored curves represent the measured binding responses of hPCSK9 when injected at concentrations of 5 nM (red) and 50 nM (green), with the global fit overlaid in black. (B) Isoaffinity plot comparing anti-PCSK9 mAbs generated from chicken immunization (olive green) with those from human phage display libraries (blue). (C) Isoaffinity plot comparing anti-PGRN mAbs generated from immunizations in chicken (olive green) and mouse (purple). The red dotted line indicates the k d limit of 5.70 × 10 −5 (1/s) that was placed on interactions which showed < 5 % signal decay within the allowed dissociation phase of 15 min, also known as the “5 % rule” (see Methods).

Article Snippet: Mouse anti-PGRN mAbs were generated via standard hybridoma technology using a single Balb/c mouse that was immunized with 50 μg recombinant hPGRN (R&D systems, 2420-PG) mixed with Gerbu adjuvant with weekly intraperitoneal (i.p.) boosts for 5 boosts total.

Techniques: Comparison, Derivative Assay, Binding Assay, Injection, Generated

Journal: mAbs

Article Title: Assessing kinetic and epitopic diversity across orthogonal monoclonal antibody generation platforms

doi: 10.1080/19420862.2015.1118596

Figure Lengend Snippet: Solution affinity determination of 2 high affinity clones obtained from chicken immunization toward their respective serum antigens. (A) Schematic representation of the KinExA assay set-up. The mAb of interest is titrated into human serum and the equilibrated mixtures are injected over beads absorption-coated with a competing mAb to capture the free antigen. The bead-captured antigen is then detected using a Dylight-labeled sandwiching mAb. (B) Global analysis of mAb C34 binding serum PCSK9. (C) Global analysis of mAb C25 binding serum PGRN. In each case, the reported apparent K D value is the best fit and 95 % confidence interval of the fit.

Article Snippet: Mouse anti-PGRN mAbs were generated via standard hybridoma technology using a single Balb/c mouse that was immunized with 50 μg recombinant hPGRN (R&D systems, 2420-PG) mixed with Gerbu adjuvant with weekly intraperitoneal (i.p.) boosts for 5 boosts total.

Techniques: Clone Assay, Injection, Labeling, Binding Assay

Journal: mAbs

Article Title: Assessing kinetic and epitopic diversity across orthogonal monoclonal antibody generation platforms

doi: 10.1080/19420862.2015.1118596

Figure Lengend Snippet: Chicken-mouse merged binning results for PGRN. (A) Heat map showing binning assignments for 32 mAbs generated from chicken immunization (olive green) and 20 mAbs generated from mouse immunization (purple). SPR-derived K D values toward human PGRN are reported, along with their cross-reaction toward mouse (m) PGRN, and their GEP subdomain assignment (just for the human-specific clones; n/d = not determined). (B) Blocking network plot, colored by mAb library, with GEP assignments indicated (determined empirically, or inferred). (C) Dendrogram of the antibody sequence lineages for the chicken mAbs alongside the binning heat map for these clones (drawn from panel A, transposed, and resorted). (D) Blocking network plot for the chicken clones, colored by bin, with GEP assignments indicated. See Table S2.

Article Snippet: Mouse anti-PGRN mAbs were generated via standard hybridoma technology using a single Balb/c mouse that was immunized with 50 μg recombinant hPGRN (R&D systems, 2420-PG) mixed with Gerbu adjuvant with weekly intraperitoneal (i.p.) boosts for 5 boosts total.

Techniques: Generated, Derivative Assay, Clone Assay, Blocking Assay, Sequencing

Journal: mAbs

Article Title: Assessing kinetic and epitopic diversity across orthogonal monoclonal antibody generation platforms

doi: 10.1080/19420862.2015.1118596

Figure Lengend Snippet: Comparison of the epitope coverage observed for anti-PGRN mAbs raised via the immunization of chickens or mouse . *Of the 14 chicken mAbs that showed human/mouse crossreactivity, 8 were mapped to a GEP subdomain (by inferring from the binning data shown in Fig. 4B ) and are included in the A – P tally, and 6 were not assigned to a GEP subdomain.

Article Snippet: Mouse anti-PGRN mAbs were generated via standard hybridoma technology using a single Balb/c mouse that was immunized with 50 μg recombinant hPGRN (R&D systems, 2420-PG) mixed with Gerbu adjuvant with weekly intraperitoneal (i.p.) boosts for 5 boosts total.

Techniques: Comparison

Journal: The Journal of Cell Biology

Article Title: Prosaposin facilitates sortilin-independent lysosomal trafficking of progranulin

doi: 10.1083/jcb.201502029

Figure Lengend Snippet: Physical interaction between PGRN and PSAP. (A) PGRN and PSAP interact when overexpressed in HEK293T cells. PSAP-V5 and FLAG-PGRN constructs were transfected into HEK293T cells as indicated. Lysates were prepared 2 d later and immunoprecipitated with anti-FLAG antibodies. (B and C) PSAP and PGRN interact with each other at endogenous levels in fibroblasts. Cell lysates (B) and conditioned medium (C) from wild-type (WT), PSAP −/− (PS−/−), and PGRN −/− (GRN−/−) fibroblasts were immunoprecipitated using rabbit anti-PGRN antibodies. The presence of PGRN and PSAP in the immunoprecipitation was detected using sheep anti–mouse PGRN and rabbit anti–mouse PSAP antibodies, respectively. Asterisk indicates IgG bands. (D) Direct interaction between PGRN and PSAP. Purified recombinant his-PSAP and Flag-PGRN proteins of indicated amounts were mixed in 100 µl PBS + 0.2% Triton X-100 for 1 h before adding anti-FLAG beads. Beads were washed after a 2-h incubation. 0.2 µg of each purified protein was loaded as input. After SDS-PAGE, the gel was stained with Krypton dye to visualize proteins. The binding ratio of PGRN–PSAP was provided based on the densitometric evaluation of PGRN and PSAP band intensities. All the results shown are the representative pictures from at least three independent experiments.

Article Snippet: Sheep anti–mouse PGRN and goat anti–human PGRN antibodies were obtained from R&D Systems.

Techniques: Construct, Transfection, Immunoprecipitation, Purification, Recombinant, Incubation, SDS Page, Staining, Binding Assay

Journal: The Journal of Cell Biology

Article Title: Prosaposin facilitates sortilin-independent lysosomal trafficking of progranulin

doi: 10.1083/jcb.201502029

Figure Lengend Snippet: PSAP is required for PGRN lysosomal targeting in fibroblasts. (A) Mislocalization of PGRN in PSAP −/− fibroblasts. Immunostaining for PGRN, LAMP1, and PSAP in fibroblasts derived from wild-type and PSAP −/− mice using sheep anti– mouse PGRN, rat anti–mouse LAMP1, and rabbit anti–mouse PSAP antibodies. PGRN mislocalization was observed in all the PSAP −/− fibroblasts examined. (B) Quantification of PGRN localization within LAMP1-positive vesicles in wild-type and PSAP −/− fibroblasts using ImageJ. Data are presented as mean ± SEM from three independent experiments. ***, P < 0.001, Student’s t test. (C) Increased PGRN secretion in PSAP −/− fibroblasts. Fibroblasts were cultured in serum-free medium for 48 h before the lysates and conditioned media (CM) were harvested. Proteins from the conditioned media were precipitated with TCA. (D) Quantification of experiments in C. PGRN levels are normalized to transferrin in the conditioned media and Gapdh in the cell lysates. Data are presented as mean ± SEM from four independent experiments. *, P < 0.05, Student’s t test; N.S., no significance. (E) Immunostaining for PGRN, PDI, and cathepsin D in fibroblasts derived from wild-type and PSAP −/− mice. Representative images from three replicated experiments are shown Bars: (main) 20 µm; (inset) 5 μm.

Article Snippet: Sheep anti–mouse PGRN and goat anti–human PGRN antibodies were obtained from R&D Systems.

Techniques: Immunostaining, Derivative Assay, Cell Culture

Journal: The Journal of Cell Biology

Article Title: Prosaposin facilitates sortilin-independent lysosomal trafficking of progranulin

doi: 10.1083/jcb.201502029

Figure Lengend Snippet: PSAP facilitates PGRN lysosomal targeting from the extracellular space . (A) PSAP facilitates PGRN lysosomal targeting from the extracellular space in fibroblasts. GRN −/− fibroblasts were treated with recombinant human his-PSAP and human FLAG-PGRN-his at a concentration of 5 µg/ml in serum-free media for 12 h as indicated. Fixed cells were stained with goat anti–human PGRN, rat anti–mouse LAMP1, and rabbit anti–human saposin B (PSAP) antibodies. Representative images from three replicated experiments are shown. Bars: (main) 20 µm; (inset) 5 μm. (B) Western blot analysis of PGRN and PSAP proteins in the uptake assay. GRN −/− fibroblasts were treated with PGRN and PSAP proteins as in A and the proteins in the lysate after 24-h uptake as well proteins in the medium before and after 24-h uptake are shown. Western blots were detected using goat anti–human PGRN and rabbit anti–human saposin B antibodies. For unknown reasons, PSAP signal is always stronger in the medium when PGRN is added together in the Western blot (although the same amount of PSAP protein was added). (C) Quantification of PGRN–PSAP levels in the medium for experiment in B. Data are presented as mean ± SEM from three independent experiments. ***, P < 0.01, Student’s t test; N.S., no significance.

Article Snippet: Sheep anti–mouse PGRN and goat anti–human PGRN antibodies were obtained from R&D Systems.

Techniques: Recombinant, Concentration Assay, Staining, Western Blot

Journal: The Journal of Cell Biology

Article Title: Prosaposin facilitates sortilin-independent lysosomal trafficking of progranulin

doi: 10.1083/jcb.201502029

Figure Lengend Snippet: Sortilin and PSAP comprise two independent and complementary pathways for PGRN lysosomal targeting. (A) Immunoblot for sortilin with lysates prepared from fibroblasts and N2a cells. Gapdh was used as a loading control. (B) Immunostaining for PGRN, LAMP1, and PSAP in fibroblasts derived from wild-type and Sort1 −/− mice. (C) Ectopic expression of sortilin in PSAP −/− fibroblasts rescues the PGRN trafficking defect. PSAP −/− fibroblasts were transfected with GFP-sortilin. 48 h after transfection, the cells were fixed and stained with sheep anti–mouse PGRN and rabbit anti–Cathepsin D. Representative images from three replicated experiments are shown. Bars: (main) 20 µm; (inset) 5 μm.

Article Snippet: Sheep anti–mouse PGRN and goat anti–human PGRN antibodies were obtained from R&D Systems.

Techniques: Western Blot, Immunostaining, Derivative Assay, Expressing, Transfection, Staining

Journal: The Journal of Cell Biology

Article Title: Prosaposin facilitates sortilin-independent lysosomal trafficking of progranulin

doi: 10.1083/jcb.201502029

Figure Lengend Snippet: M6PR and LRP1 mediate PGRN–PSAP lysosomal trafficking. (A) Both PSAP and PGRN are mislocalized in M6PR-deficient fibroblasts. Fibroblasts infected with lentiviruses harboring guide RNA against M6PR and Cas9 were selected with puromycin and fixed and stained with goat anti–mouse PSAP or sheep anti–PGRN antibodies as indicated together with rat anti-LAMP1 and rabbit anti-M6PR antibodies. Two neighboring cells with (a) or without (b) M6PR expression are shown. (B and C) Quantification of PSAP (B) and PGRN (C) localization within LAMP1-positive vesicles in control and M6PR −/− fibroblasts as shown in A using ImageJ. Data are given as mean ± SEM from three independent experiments: **, P < 0.01; Student’s t test. (D and E) M6PR is required for PSAP-mediated PGRN lysosomal targeting from the extracellular space. Fibroblasts infected with lentiviruses harboring guide RNA against M6PR and Cas9 were selected with puromycin for a week and treated with recombinant human his-PSAP and human FLAG-PGRN-his proteins (5 µg/ml) in serum-free medium for 12 h, and then stained with hPGRN, LAMP1, and M6PR antibodies. Two neighboring cells with (a) or without (b) M6PR expression are shown. Intensities of endocytosed PGRN were quantified using ImageJ. Data are presented as mean ± SEM from three independent experiments: ***, P < 0.001, Student’s t test. (F and G) LRP1 is also critical for PGRN–PSAP uptake in fibroblasts. Fibroblasts infected with lentiviruses harboring guide RNA against LRP1 and Cas9 were selected with puromycin for 1 wk and treated as in D. Data are presented as mean ± SEM from three independent experiments: ***, P < 0.001, Student’s t test. Bars: (main) 20 µm; (inset) 5 μm.

Article Snippet: Sheep anti–mouse PGRN and goat anti–human PGRN antibodies were obtained from R&D Systems.

Techniques: Infection, Staining, Expressing, Recombinant

Journal: Stem Cells Translational Medicine

Article Title: Progranulin, a Major Secreted Protein of Mouse Adipose-Derived Stem Cells, Inhibits Light-Induced Retinal Degeneration

doi: 10.5966/sctm.2013-0020

Figure Lengend Snippet: Progranulin suppressed H2O2- or light-induced cultured photoreceptor cell death. (A, B): Quantitative determination of progranulin in ASC-CM by Western blot. (A): Representative immunoblots showing progranulin protein levels in ASC-CM and recombinant mouse progranulin. The recombinant progranulin was expressed in a mouse myeloma cell line, and post-translational modifications such as glycosylation may contribute to various molecular weights. (B): Standard curve was generated from the density of mouse recombinant progranulin. (C, E): Representative fluorescence microscopy images showing nuclear staining for Hoechst 33342 and PI after 27 hours of H2O2 (0.3 mM) treatment (C) or 24 hours of light exposure (E). (D, F): The number of cells exhibiting PI fluorescence was counted, and positive cells were expressed as the percentage of PI- to Hoechst 33342-positive cells. The number of PI-positive cells increased after H2O2 treatment or light exposure. Progranulin significantly reduced cell death in a concentration-dependent manner. Scale bars = 50 µm. Data are shown as mean ± SEM (n = 6 or n = 9). *, p < .05 versus vehicle; **, p < .01; ##, p < .01 versus control. Abbreviations: ASC-CM, adipose-derived stem cell-conditioned medium; Con, control; H2O2, hydrogen peroxide; PI, propidium iodide; Veh, vehicle.

Article Snippet: For immunoblotting, the following primary antibodies were used: monoclonal anti-mouse progranulin antibody (R&D Systems), anti-β-actin, (Sigma-Aldrich), anti-ERK1/2, anti-phosphorylated ERK1/2, anti-HGF receptor (MET), anti-phosphorylated MET (Cell Signaling Technology, Danvers, MA, http://www.cellsignal.com ), anti-cAMP response element binding protein (Cell Signaling Technology), and anti-phosphorylated CREB (Santa Cruz Biotechnology, Inc., Santa Cruz, CA, http://www.scbt.com ).

Techniques: Cell Culture, Western Blot, Recombinant, Generated, Fluorescence, Microscopy, Staining, Concentration Assay, Derivative Assay

Journal: Stem Cells Translational Medicine

Article Title: Progranulin, a Major Secreted Protein of Mouse Adipose-Derived Stem Cells, Inhibits Light-Induced Retinal Degeneration

doi: 10.5966/sctm.2013-0020

Figure Lengend Snippet: Progranulin reduced retinal damage induced by exposure to light in mice. (A): Representative photographs of hematoxylin and eosin staining are as follows: nontreated group (Aa), light exposure (8,000 lx) plus vehicle-treated group (Ab), and light exposure plus progranulin-treated (200 ng per eye) group (Ac) at 5 days after light exposure. (B): ONL thickness was measured at 5 days after light exposure. The ONL was measured at 240-μm intervals from the optic disc. Scale bar = 25 µm. Data are shown as mean ± SEM (n = 7 or n = 8). *, p < .05 versus the light exposure plus vehicle-treated group (vehicle). Abbreviation: ONL, outer nuclear layer.

Article Snippet: For immunoblotting, the following primary antibodies were used: monoclonal anti-mouse progranulin antibody (R&D Systems), anti-β-actin, (Sigma-Aldrich), anti-ERK1/2, anti-phosphorylated ERK1/2, anti-HGF receptor (MET), anti-phosphorylated MET (Cell Signaling Technology, Danvers, MA, http://www.cellsignal.com ), anti-cAMP response element binding protein (Cell Signaling Technology), and anti-phosphorylated CREB (Santa Cruz Biotechnology, Inc., Santa Cruz, CA, http://www.scbt.com ).

Techniques: Staining

Journal: Stem Cells Translational Medicine

Article Title: Progranulin, a Major Secreted Protein of Mouse Adipose-Derived Stem Cells, Inhibits Light-Induced Retinal Degeneration

doi: 10.5966/sctm.2013-0020

Figure Lengend Snippet: Progranulin exerted a photoreceptor protective effect through the protein kinase C pathway. Representative band images show immunoreactivity against pERK, total ERK, pCREB, and total CREB (A) and phosphorylated HGFR and total HGFR (D) after progranulin treatment (500 ng/ml) at 5, 10, 30, and 60 minutes. (B, E): Quantitative analysis of band densities. Data are shown as mean ± SEM (n = 3). (C): Progranulin (500 ng/ml), U0126 (1,000 nM), H-87 (500 nM), and Gö 6976 (500 nM) were added to 661W cells, and the cells were irradiated with white light (2,500 lx) for 24 hours. Cell death rate was calculated as described in Materials and Methods. Data are shown as mean ± SEM (n = 6). *, p < .05 versus vehicle; **, p < .01. Abbreviations: CREB, cAMP response element binding protein; HGFR, hepatocyte growth factor receptor; n.s., not significant; P, progranulin; pCREB, phosphorylated CREB; pERK, phosphorylated ERK; V, vehicle.

Article Snippet: For immunoblotting, the following primary antibodies were used: monoclonal anti-mouse progranulin antibody (R&D Systems), anti-β-actin, (Sigma-Aldrich), anti-ERK1/2, anti-phosphorylated ERK1/2, anti-HGF receptor (MET), anti-phosphorylated MET (Cell Signaling Technology, Danvers, MA, http://www.cellsignal.com ), anti-cAMP response element binding protein (Cell Signaling Technology), and anti-phosphorylated CREB (Santa Cruz Biotechnology, Inc., Santa Cruz, CA, http://www.scbt.com ).

Techniques: Irradiation, Binding Assay