mouse antinephrin  (Santa Cruz Biotechnology)

Journal: Kidney360

Article Title: Dynll1-PI31 Interaction Enhances Proteolysis Through the Proteasome, Representing a Novel Therapeutic Target for INF2-Related FSGS

doi: 10.34067/KID.0000000659

Figure Lengend Snippet: Antibodies and assay kits

Article Snippet: Mouse antinephrin (G-8) , Santa Cruz , sc-376522, AF488, AF594.

Techniques: Membrane, Staining, Activity Assay

Journal: Kidney360

Article Title: Dynein-Mediated Trafficking: A New Mechanism of Diabetic Podocytopathy

doi: 10.34067/KID.0006852022

Figure Lengend Snippet: Enhanced dynein-mediated postendocytic trafficking of nephrin in hyperglycemic conditions. (A) In vitro nephrin trafficking assay in mouse podocytes using antibody-mediated cross-linking of nephrin: Nephrin molecules expressed on the podocyte surface were cross-linked by an antinephrin primary antibody and Alexa Fluro 594–labeled secondary antibody, which triggered nephrin internalization and recruitment of trafficking adapters. As demonstrated by Co-IP ((B) by measuring dynein components in nephrin pulldown) and an immunofluorescence colocalization assay ((C) cross-linked nephrin labeled in red by Alexa Fluor 594, Dynll1, and DCTN1 labeled in green by Alexa Fluor 488), HG treatment (HG, 30 mM) increased the recruitment of Dynll1, DCTN1, and HDAC6 to cross-linked nephrin, compared with NG (5.5 mM). These changes could be diminished by inhibiting dynein activity (using Ciliobrevin D, 50 µM) (n=3, *P<0.05 versus HG+0.3% DMSO) or by knocking down Dynll1 in podocytes (n=3, ^P<0.05 versus HG+control siRNA).

Article Snippet: Nephrin Trafficking Model and Time-Lapse Imaging As shown in the schematics in Figure A, cells transfected with the pcDNA3-nephrin plasmid (gift from Dr. Holzman ) were incubated with a mouse antinephrin antibody recognizing the extracellular domain (Santa Cruz #sc-376522), followed by an Alexa Fluor 594-conjugated donkey anti-mouse IgG (Thermo fisher #A32744) to trigger cross-linking and internalization of nephrin.

Techniques: In Vitro, Labeling, Co-Immunoprecipitation Assay, Immunofluorescence, Activity Assay, Control

Journal: Kidney360

Article Title: Dynein-Mediated Trafficking: A New Mechanism of Diabetic Podocytopathy

doi: 10.34067/KID.0006852022

Figure Lengend Snippet: In vivo evidence of dynein-related mistrafficking and degradation of nephrin. (A) IF staining showed that overexpressed Dynll1 colocalizes with nephrin in STZ-induced mouse diabetic podocytopathy. The amount of Dynll1 colocalized with nephrin in nonsclerotic glomeruli was quantified using the Colo2 plugin of Fiji software using the Manders overlap coefficient for comparison. *P<0.05 versus vehicle control, n=6 mice×3 glomeruli per mouse. (B) The recruitments of Dynll1 and DCTN1 were examined in nephrin pulldown and were expressed as the ratio to total nephrin, reflecting the involvement of dynein in nephrin trafficking in vivo. Nephrin protein targeted for ubiquitin-mediated degradation was examined by measuring ubiquitinated nephrin (IP with antinephrin and IB with anti-ubiquitin). Nephrin localized on the podocyte surface was measured by streptavidin IP after the in vivo surface biotinylation assay, expressed as the percentage of total nephrin for comparison. *P<0.05 versus vehicle control, n=6. IP, immunoprecipitation.

Article Snippet: Nephrin Trafficking Model and Time-Lapse Imaging As shown in the schematics in Figure A, cells transfected with the pcDNA3-nephrin plasmid (gift from Dr. Holzman ) were incubated with a mouse antinephrin antibody recognizing the extracellular domain (Santa Cruz #sc-376522), followed by an Alexa Fluor 594-conjugated donkey anti-mouse IgG (Thermo fisher #A32744) to trigger cross-linking and internalization of nephrin.

Techniques: In Vivo, Staining, Software, Comparison, Control, Ubiquitin Proteomics, Surface Biotinylation Assay, Immunoprecipitation

Journal: Kidney360

Article Title: Dynein-Mediated Trafficking: A New Mechanism of Diabetic Podocytopathy

doi: 10.34067/KID.0006852022

Figure Lengend Snippet: Antibodies and fluorescent probes (immunofluorescent, Western blotting, immunoprecipitation]

Article Snippet: Nephrin Trafficking Model and Time-Lapse Imaging As shown in the schematics in Figure A, cells transfected with the pcDNA3-nephrin plasmid (gift from Dr. Holzman ) were incubated with a mouse antinephrin antibody recognizing the extracellular domain (Santa Cruz #sc-376522), followed by an Alexa Fluor 594-conjugated donkey anti-mouse IgG (Thermo fisher #A32744) to trigger cross-linking and internalization of nephrin.

Techniques: Western Blot, Immunoprecipitation

Journal: Kidney360

Article Title: Dynein-Mediated Trafficking: A New Mechanism of Diabetic Podocytopathy

doi: 10.34067/KID.0006852022

Figure Lengend Snippet: Antibodies and fluorescent probes (immunofluorescent, Western blotting, immunoprecipitation]

Article Snippet: Table 2 Antibody Dilution Company Cat# Mouse antinephrin (G-8) IF: 1:100 Santa Cruz sc-376522 Mouse antinephrin conjugated to agarose IP: 2 μg/400 μl lysate Santa Cruz sc-376522 AC Normal mouse IgG conjugated to agarose IP: 2 μg/400 μl lysate Santa Cruz sc-2343 Rabbit antinephrin WB: 1:1000 Invitrogen PA5-91907 Sheep anti-DCTN1 WB: 1:1000 IF: 1:100 R&D Systems AF6657 Mouse anti-Dynll1 IF: 1:100 Santa Cruz sc-136287 Rabbit anti-Dynll1 WB: 1:1000 IF: 1:100 Thermo Fisher PA5-97920 Rabbit anti-INF2 IF: 1:100 Bethyl Lab A303-427A Rabbit anti-HDAC6 WB: 1:1000 Novus NBP1-78981 Donkey anti-mouse (Alexa Fluor 488) IF: 1:200 Thermo Fisher A32766 Donkey anti-rabbit (Alexa Fluor 488) IF: 1:200 Thermo Fisher A32790 Donkey anti-mouse (Alexa Fluor 594) IF: 1:200 Thermo Fisher A32744 Donkey anti-sheep (Alexa Fluor 488) IF: 1:200 Thermo Fisher A-11015 Mouse anti-rabbit IgG-HRP WB: 1:5000 Santa Cruz sc-2357 Rabbit anti-sheep IgG-HRP WB: 1:5000 Thermo Fisher 31480 Biotin mouse anti-ubiquitin WB: 1:1000 Invitrogen 13-6078-82 Mouse anti β-actin-HRP WB: 1:5000 Santa Cruz sc-47778 Mouse anti-WT1 IF: 1:100 Novus NBP24460700 Mouse anti-acetylated α-tubulin IF: 1:200 Santa Cruz sc-23950 Acti-stain 488 phalloidin Cytoskeleton PHDG1-A ViaFluor microtubule stains (488) Biotium 70062 Open in a separate window IF, immunofluorescent; IP, immunoprecipitation; WB, Western blotting; INF2, inverted formin 2; HDAC6, histone deacetylase 6; HRP, horseradish peroxidase.

Techniques: Western Blot, Immunoprecipitation