recombinant human norrin protein  (R&D Systems)

Journal: International Journal of Molecular Sciences

Article Title: Development of Norrin-Based Protein Therapeutic for Activation of Norrin-Wnt Signaling in Human Retinal Endothelial Cells

doi: 10.3390/ijms262311340

Figure Lengend Snippet: Expression of tag-less Norrin K86P in BL21(DE3) cell inclusion body protein fractions. DISC-PAGE protein gel of inclusion body (IB) preparations from four separate batches are shown. IB material was dissolved and reduced by heating (100 °C × 10 min) in SDS sample buffer with 2-mercaptoethanol. Some dimeric and tetrameric bands can be formed during this treatment. Two different loading volumes (1 µL and 5 µL) were used for each of the four batches shown. Gels were stained with colloidal Coomassie blue. IB protein content was >98% recombinant Norrin K86P .

Article Snippet: The cells were weaned to media containing no Hydrocortisone Hemisuccinate prior to stimulation with Norrin K86P or rh Norrin (R&D Systems, Minneapolis, MN, USA; 3014-NR).

Techniques: Expressing, Staining, Recombinant

Journal: International Journal of Molecular Sciences

Article Title: Development of Norrin-Based Protein Therapeutic for Activation of Norrin-Wnt Signaling in Human Retinal Endothelial Cells

doi: 10.3390/ijms262311340

Figure Lengend Snippet: Fundus and fluorescein angiography images before and after injection of Norrin K86P . Long Evans rats were imaged pre injection and 3 weeks post injection of 250 ng Norrin K86P . An example of the same Norin K86P -treated eye (OS) and vehicle-treated contralateral eye (OD) are shown. Brightfield fundus images are shown for each eye with their corresponding fluoresceine angiography image below. Angiography shows the perfused microvasculature of the neural retina (green). No impact on the retinal vasculature was noted for any eyes, whether Norin K86P - or vehicle-injected, across all rats tested. (N = 4). The same retinas were also analyzed for retinal thickness using SD-OCT.

Article Snippet: The cells were weaned to media containing no Hydrocortisone Hemisuccinate prior to stimulation with Norrin K86P or rh Norrin (R&D Systems, Minneapolis, MN, USA; 3014-NR).

Techniques: Injection

Journal: International Journal of Molecular Sciences

Article Title: Development of Norrin-Based Protein Therapeutic for Activation of Norrin-Wnt Signaling in Human Retinal Endothelial Cells

doi: 10.3390/ijms262311340

Figure Lengend Snippet: Retinal thickness before and after intraocular injection of Norrin K86P . Long Evans rats received intraocular injections of vehicle (Veh) OD and 250 ng of Norrin K86P (Nor) OS. Average retinal thickness was calculated from 24 measurements around the optic disk for each retina, before injection and then again three weeks post injection. Thickness was measured between the inner limiting membrane (ILM) and outer limiting membrane (OLM) of the neural retina. The retinal thickness values (mm) averaged from four different rats are shown; bars show standard deviation.

Article Snippet: The cells were weaned to media containing no Hydrocortisone Hemisuccinate prior to stimulation with Norrin K86P or rh Norrin (R&D Systems, Minneapolis, MN, USA; 3014-NR).

Techniques: Injection, Membrane, Standard Deviation

Journal: International Journal of Molecular Sciences

Article Title: Development of Norrin-Based Protein Therapeutic for Activation of Norrin-Wnt Signaling in Human Retinal Endothelial Cells

doi: 10.3390/ijms262311340

Figure Lengend Snippet: Mixed Rod–Cone ERG response of Long Evans rats. Long Evans rats received intraocular injections of vehicle (Veh) OD and 250 ng of Norrin K86P (Nor) OS. ( A ) Examples of Rod and mixed Rod–Cone ERG recordings from vehicle and contralateral Norrin K86P -injected eyes, 3 weeks post-injection. The left vertical marker indicates the light pulse flash at 0 milliseconds (ms). ( B ) Mixed Rod–Cone ERGs were measured, comparing vehicle-injected (OD) and Norrin-injected eyes, after dark adaptation. Average magnitudes of A-wave and B-wave (uV) are shown, using full-field white-light stimulation of 3.0 cd-sec/m 2 . Bars show standard deviation (N = 4 rats). No inhibition of Cone–Rod ERG response was detected in Norrin-treated versus vehicle-treated eyes.

Article Snippet: The cells were weaned to media containing no Hydrocortisone Hemisuccinate prior to stimulation with Norrin K86P or rh Norrin (R&D Systems, Minneapolis, MN, USA; 3014-NR).

Techniques: Injection, Marker, Standard Deviation, Inhibition

Journal: International Journal of Molecular Sciences

Article Title: Development of Norrin-Based Protein Therapeutic for Activation of Norrin-Wnt Signaling in Human Retinal Endothelial Cells

doi: 10.3390/ijms262311340

Figure Lengend Snippet: Norrin-based protein sequences inserted into the pD454-MBP vector. Inserted sequences were added in frame following the MBP sequence. The resulting proteins were MBP N-terminal fusion proteins. ( A ) Nor-2 contained the mature human Norrin K86P , sequence underlined. The HRV3C protease cleavage site is shown in bold font. ( B ) Nor-w-Linker included extra linker sequences to improve the steric exposure of the HRV3C cleavage site. Three rigid helical linkers (blue font) and three flexible linkers (red font) are indicated. Two polypeptides identified by mass spectroscopy after electrophoresis of a Norrin-sized band (15 kDa) obtained after HRV3C protease treatment are highlighted with yellow.

Article Snippet: The cells were weaned to media containing no Hydrocortisone Hemisuccinate prior to stimulation with Norrin K86P or rh Norrin (R&D Systems, Minneapolis, MN, USA; 3014-NR).

Techniques: Plasmid Preparation, Sequencing, Mass Spectrometry, Electrophoresis

Journal: bioRxiv

Article Title: A recombinant norrin-derived growth factor retains the complete multifunctionality of human norrin

doi: 10.1101/2025.10.15.682582

Figure Lengend Snippet: Norrin activates the β-Catenin pathway through both the FZD4 receptor in endothelial cells (1) and the LGR4 receptor in neural cells (2). In addition, norrin inhibits TGF-beta and BMP-2/4 signaling (3).

Article Snippet: An example 4-PL curve fit ( ) comparing Noregen to human recombinant norrin (R&D Systems, Minneapolis, MN) demonstrates that Noregen has an equal, if not superior, FZD4 receptor binding efficacy compared to that of norrin.

Techniques:

Journal: bioRxiv

Article Title: A recombinant norrin-derived growth factor retains the complete multifunctionality of human norrin

doi: 10.1101/2025.10.15.682582

Figure Lengend Snippet: (A) Noregen FZD4 receptor binding activity compared to Norrin, EC 50 19 ng/mL & 34 ng/mL respectively. (B) Noregen dose response curve for TCF/LEF reporter activation in HEK-293 cells (EC 50 11 ng/mL). (C) Noregen induced Axin-2 gene expression in HRMECs. Values are means +/-SD; *P<0.05, **P<0.01, ***P<0.001 & ****P<0.0001.

Article Snippet: An example 4-PL curve fit ( ) comparing Noregen to human recombinant norrin (R&D Systems, Minneapolis, MN) demonstrates that Noregen has an equal, if not superior, FZD4 receptor binding efficacy compared to that of norrin.

Techniques: Binding Assay, Activity Assay, Activation Assay, Gene Expression

Journal: bioRxiv

Article Title: A recombinant norrin-derived growth factor retains the complete multifunctionality of human norrin

doi: 10.1101/2025.10.15.682582

Figure Lengend Snippet: (A) Noregen significantly increases HRMEC proliferation as seen by a drop in capacitance when treated at 20 and 500 ng/mL. (B) Norrin increases proliferation to a similar extent as Noregen at the higher 500 ng/mL treatment level, but to a lesser extent at 20 ng/mL. (C) Comparison of Noregen and norrin treatments at 20 ng/mL. An earlier drop in capacitance indicates faster growth in Noregen treated cells.

Article Snippet: An example 4-PL curve fit ( ) comparing Noregen to human recombinant norrin (R&D Systems, Minneapolis, MN) demonstrates that Noregen has an equal, if not superior, FZD4 receptor binding efficacy compared to that of norrin.

Techniques: Comparison

Journal: bioRxiv

Article Title: A recombinant norrin-derived growth factor retains the complete multifunctionality of human norrin

doi: 10.1101/2025.10.15.682582

Figure Lengend Snippet: (A) Reduction in normalized avascular area [AVA to Total Area ratio] in Noregen & norrin injected eyes. The [AVA to Total Area ratio] in injected eyes was normalized to fellow un-injected eyes. Noregen levels were 2, 20 or 200 ng. Values are means +/-SEM; *P<0.05, **P<0.01. (B) Dose dependent reduction in normalized avascular area seen when graphing in comparison to averaged Vehicle from combined cohorts. (C) Example retinal flatmount images with AVA highlighted in white. (D) Confocal images of superficial (S), intermediate (I) & deep (D) vascular beds in Noregen(200 ng/eye) and vehicle injected OIR eyes.

Article Snippet: An example 4-PL curve fit ( ) comparing Noregen to human recombinant norrin (R&D Systems, Minneapolis, MN) demonstrates that Noregen has an equal, if not superior, FZD4 receptor binding efficacy compared to that of norrin.

Techniques: Injection, Comparison

Journal: bioRxiv

Article Title: A recombinant norrin-derived growth factor retains the complete multifunctionality of human norrin

doi: 10.1101/2025.10.15.682582

Figure Lengend Snippet: (A) Diagram illustrating TGF-ß1 and BMP-2 pathways. Norrin inhibits signaling via SMAD7 induction, thereby inhibiting pathway activation. (B) Noregen reduction in basal level and TGF-ß1 induced SMAD reporter activation. Values are means +/-SD; *P<0.05, **P<0.01, ***P<0.001. (C) ZO-1 immunostaining of HRMECs treated with TGF-ß1 or BMP-2 alone (translocation of ZO-1 to cytosol) or in combination with Noregen (restoration of ZO-1 at plasma membrane).

Article Snippet: An example 4-PL curve fit ( ) comparing Noregen to human recombinant norrin (R&D Systems, Minneapolis, MN) demonstrates that Noregen has an equal, if not superior, FZD4 receptor binding efficacy compared to that of norrin.

Techniques: Activation Assay, Immunostaining, Translocation Assay, Clinical Proteomics, Membrane