biotin labeled p cadherin antibody  (Bioss)

Journal: Journal of the American Society of Nephrology : JASN

Article Title: ADAM10-Mediated Ectodomain Shedding Is an Essential Driver of Podocyte Damage

doi: 10.1681/ASN.2020081213

Figure Lengend Snippet: ADAM10-mediated N-cadherin shedding is essential for APN antibody–induced podocyte injury. (A) Quantification of podocyte number per glomerular tuft area in naive and APN-treated ADAM10Δpod and control littermates. Graph exhibits violin plot of pooled data from four independent experiments (n≥12 per group). (B) Adhesion assay demonstrates APN antibody–induced murine podocyte detachment from the culture dish, which could be suppressed by an ADAM10 inhibitor. The ADAM10 activator ionomycin served as the positive control. One representative out of three individual experiments is shown, each with six replicates per condition. (C) APN antibody (ab) reduces N-cadherin cell-surface levels from murine podocytes, which was rescued by administration of ADAM10 inhibitor GI254023X. The cell-surface protein transferrin receptor and soluble glyceraldehyde-3-phosphate dehydrogenase (GAPDH) served as control for loading and purity of cell-surface extracts. One representative out of three experiments is shown. (D) N-cadherin shedding is increased in response to APN antibody in cultured murine podocytes. ADAM10 inhibition with GI254023X suppressed generation of N-cadherin CTF (n=4). In parallel, podocytes were treated with 1 µM DAPT to stabilize the generated N-cadherin CTF. Splice variant (SV) based on the murine protein information at uniprot.org/uniprot/P15116#ptm_processing and uniprot.org/uniprot/D3YYT0#sequences. *P≤0.05, **P≤0.01, ***P≤0.001, ****P≤0.0001.

Article Snippet: Antibodies The following antibodies were used for the study: rat anti-ADAM10 (IF human and naive mice, 1:100; immunogold electron microscopy [EM], 1:25; mAb946 R&D Systems), rabbit anti-ADAM10 (IF human and diseased mice, 1:100; WB, 1:1000; Abcam), rabbit anti-ADAM10 (IF diseased mice, 1:100; WB, 1:1000; Pineda), rabbit anti-ADAM10 (IF diseased mice, 1:100; WB, 1:1000; GTX63486 GenTex), rabbit anti–N-cadherin (WB, 1:1000; IF, 1:200; Abcam), mouse anti–N-cadherin (IF cells, 1:100; 610921BD Bioscience), rabbit anti–P-cadherin (IF, 1:100; bs-1159r Bioss), rabbit anti– β -catenin (WB, 1:1000; IF, 1:100; Cell Signaling), rabbit anti–phospho- β -catenin (WB, 1:1000; IF, 1:100; Millipore), guinea pig anti-nephrin (IF, 1:200; BP5030 Acris), rabbit anti-synaptopodin (IF 1:200; Sc-50459 Santa Cruz), mouse anti– β -actin (WB, 1:3000; A2066 Sigma), rabbit anti-laminin (IF, 1:500; L9393 Sigma), Cy5 donkey anti-sheep and Cy5 donkey anti-mouse (IF, 1:200; Jackson ImmunoResearch Laboratories, West Grove, PA), PE-podoplanin (FACS sort, 1:200; clone 8.1.1.

Techniques: Cell Adhesion Assay, Positive Control, Cell Culture, Inhibition, Generated, Variant Assay

Journal: Journal of the American Society of Nephrology : JASN

Article Title: ADAM10-Mediated Ectodomain Shedding Is an Essential Driver of Podocyte Damage

doi: 10.1681/ASN.2020081213

Figure Lengend Snippet: ADAM10 deficiency results in enhanced cadherin levels on podocytes in APN antibody–induced podocyte injury. APN was induced in ADAM10Δpod and control (ctrl) littermate mice, and kidneys were analyzed on day 14. (A) Immunoblot for the cell-cell adhesion molecule N-cadherin (N-cad) levels in isolated glomeruli. Graph exhibits densitometric quantification of n=8 mice per genotype; pooled data from two independent experiments. *P≤0.05. The levels of α-actinin 4 within respective glomerular lysates suggest comparable amounts of podocytes. Splice variant (SV) based on the murine protein information at uniprot.org/uniprot/P15116#ptm_processing and uniprot.org/uniprot/D3YYT0#sequences. (B) Kidneys of mice with comparable mild proteinuria were stained for N-cadherin (green) in relation to the SD protein nephrin (red), and DNA (Hoechst, blue). Note the enhanced expression of N-cadherin in ADAM10Δpod podocytes (p) in comparison to control podocytes. White arrows point toward N-cadherin in presumably primary podocyte processes. (C) Representative confocal images demonstrate enhanced P-cadherin (green) in ADAM10Δpod podocytes in comparison to control podocytes; lectin wheat germ agglutinin (red) visualizes the GFB; DNA (Draq5) shown in blue. Care was taken to use N- and P-cadherin antibodies (Abs) raised in rabbit, so that secondary antibodies needed for visualization would not crossreact with the injected sheep IgG and the intrinsic mouse IgG. (D) Representative confocal images demonstrate podocyte N-cadherin localization in patients with MN and in anti-GBM GN in relation to nephrin (red) and DNA (Hoechst; blue). White arrows represent N-cadherin expression at the podocyte aspect of the GFB, especially in areas where nephrin is absent.

Article Snippet: Antibodies The following antibodies were used for the study: rat anti-ADAM10 (IF human and naive mice, 1:100; immunogold electron microscopy [EM], 1:25; mAb946 R&D Systems), rabbit anti-ADAM10 (IF human and diseased mice, 1:100; WB, 1:1000; Abcam), rabbit anti-ADAM10 (IF diseased mice, 1:100; WB, 1:1000; Pineda), rabbit anti-ADAM10 (IF diseased mice, 1:100; WB, 1:1000; GTX63486 GenTex), rabbit anti–N-cadherin (WB, 1:1000; IF, 1:200; Abcam), mouse anti–N-cadherin (IF cells, 1:100; 610921BD Bioscience), rabbit anti–P-cadherin (IF, 1:100; bs-1159r Bioss), rabbit anti– β -catenin (WB, 1:1000; IF, 1:100; Cell Signaling), rabbit anti–phospho- β -catenin (WB, 1:1000; IF, 1:100; Millipore), guinea pig anti-nephrin (IF, 1:200; BP5030 Acris), rabbit anti-synaptopodin (IF 1:200; Sc-50459 Santa Cruz), mouse anti– β -actin (WB, 1:3000; A2066 Sigma), rabbit anti-laminin (IF, 1:500; L9393 Sigma), Cy5 donkey anti-sheep and Cy5 donkey anti-mouse (IF, 1:200; Jackson ImmunoResearch Laboratories, West Grove, PA), PE-podoplanin (FACS sort, 1:200; clone 8.1.1.

Techniques: Western Blot, Isolation, Variant Assay, Staining, Expressing, Injection

Journal: Journal of the American Society of Nephrology : JASN

Article Title: ADAM10-Mediated Ectodomain Shedding Is an Essential Driver of Podocyte Damage

doi: 10.1681/ASN.2020081213

Figure Lengend Snippet: β-Catenin/Wnt signaling pathway is not activated in ADAM10Δpod mice exposed to APN antibodies. APN was induced in ADAM10Δpod and control littermate mice, and kidneys were analyzed on day 14. (A) Immunoblot for β-catenin levels in isolated glomeruli. Graph exhibits densitometric quantification of n=7 mice per genotype; pooled data from two independent experiments. *P≤0.05. (B) Kidneys of mice with comparable, mild proteinuria were stained for β-catenin (green) in relation to the SD protein nephrin (red), and DNA (Hoechst; blue). Note the enhanced β-catenin signal in nuclei (white arrows) and cytoplasm of podocytes (p) and in endothelial cells (red arrow). (C) qRT-PCR analyses of Wnt response genes in isolated glomeruli. Values are depicted as relative expression to naive control (ctrl) littermates after normalization to 18S as housekeeper gene; n=3–14 mice per genotype; pooled data from two independent experiments. *P≤0.05 to control naive, § P≤0.05 and §§ P≤0.01 to control and APN antibodies (Abs). (D) Scheme depicting proposed role of ADAM10 in podocytes in antibody-mediated podocyte injury. (1) Following a prominent embryonic expression of cell-cell adhesion molecules, such as P- and N-cadherin, in podocyte progenitors, healthy mature podocytes have low levels of cadherins at processes. (2) In the course of antibody-mediated injury, ADAM10 expression is enhanced. ADAM10 activity results in ectodomain shedding of cadherins. Cadherin cleavage results in a dissociation of β-catenin from the cytoplasmic cadherin domain and to a redistribution of β-catenin to the cytoplasm and nuclear compartment, where it initiates the transcription of Wnt response genes. Ultimately, this results in decreased cell-cell adhesion and proteinuria. (3) As a consequence of loss of ADAM10 activity, cadherin levels are stabilized at podocyte processes, after antibody-mediated injury. β-Catenin remains tethered to the cytoplasmic cadherin domain, and the Wnt/β-catenin signaling pathway is not activated. Podocyte adhesion is ameliorated, resulting in attenuated proteinuria. ADAM10Δpod, podocyte-specific ADAM10 deletion.

Article Snippet: Antibodies The following antibodies were used for the study: rat anti-ADAM10 (IF human and naive mice, 1:100; immunogold electron microscopy [EM], 1:25; mAb946 R&D Systems), rabbit anti-ADAM10 (IF human and diseased mice, 1:100; WB, 1:1000; Abcam), rabbit anti-ADAM10 (IF diseased mice, 1:100; WB, 1:1000; Pineda), rabbit anti-ADAM10 (IF diseased mice, 1:100; WB, 1:1000; GTX63486 GenTex), rabbit anti–N-cadherin (WB, 1:1000; IF, 1:200; Abcam), mouse anti–N-cadherin (IF cells, 1:100; 610921BD Bioscience), rabbit anti–P-cadherin (IF, 1:100; bs-1159r Bioss), rabbit anti– β -catenin (WB, 1:1000; IF, 1:100; Cell Signaling), rabbit anti–phospho- β -catenin (WB, 1:1000; IF, 1:100; Millipore), guinea pig anti-nephrin (IF, 1:200; BP5030 Acris), rabbit anti-synaptopodin (IF 1:200; Sc-50459 Santa Cruz), mouse anti– β -actin (WB, 1:3000; A2066 Sigma), rabbit anti-laminin (IF, 1:500; L9393 Sigma), Cy5 donkey anti-sheep and Cy5 donkey anti-mouse (IF, 1:200; Jackson ImmunoResearch Laboratories, West Grove, PA), PE-podoplanin (FACS sort, 1:200; clone 8.1.1.

Techniques: Western Blot, Isolation, Staining, Quantitative RT-PCR, Expressing, Activity Assay

Journal: Molecular & Cellular Proteomics : MCP

Article Title: Integrated Analysis of Quantitative Proteome and Transcriptional Profiles Reveals the Dynamic Function of Maternally Expressed Proteins After Parthenogenetic Activation of Buffalo Oocyte *

doi: 10.1074/mcp.RA118.000556

Figure Lengend Snippet: Up-regulated proteins involved in both tight junction and gap junction selected for further study

Article Snippet: The following commercially available antibodies were used as primary antibodies: anti-beta-III tubulin mouse monoclonal antibody (ab7751; Abcam, Cambridge, MA), anti-alpha 1 catenin rabbit polyclonal antibody (ab6299; Abcam), anti-MEK1 rabbit polyclonal antibody (ab32091; Abcam), anti-actin (loading control) rabbit polyclonal antibody (bs-17654R; Bioss Biotechnology Inc., Beijing, China), and anti-P-cadherin rabbit polyclonal antibody (bs-1159R; Bioss Biotechnology Inc., Beijing, China).

Techniques: