Human Dystroglycan Antibody

Detection of Human Dystroglycan by Western Blot. Western blot shows lysates of MCF-7 human breast cancer cell line, SH-SY5Y human neuroblastoma cell line, human muscle tissue, and human placenta tissue. PVDF membrane was probed with 1 µg/mL of Sheep Anti-Human Dystroglycan Antigen Affinity-purified Polyclonal Antibody (Catalog # AF6868) followed by HRP-conjugated Anti-Sheep IgG Secondary Antibody (Catalog # HAF016). Specific bands were detected for a-Dystroglycan at approximately 100-160 kDa (as indicated) and beta -Dystroglycan at approximately 42-44 kDa (as indicated). This experiment was conducted under reducing conditions and using Immunoblot Buffer Group 1.

Dystroglycan in Human Skeletal Muscle. Dystroglycan was detected in immersion fixed paraffin-embedded sections of human skeletal muscle using Sheep Anti-Human Dystroglycan Antigen Affinity-purified Polyclonal Antibody (Catalog # AF6868) at 3 µg/mL overnight at 4 °C. Before incubation with the primary antibody, tissue was subjected to heat-induced epitope retrieval using Antigen Retrieval Reagent-Basic (Catalog # CTS013). Tissue was stained using the Anti-Sheep HRP-DAB Cell & Tissue Staining Kit (brown; Catalog # CTS019) and counterstained with hematoxylin (blue). Specific staining was localized to basement membrane. View our protocol for Chromogenic IHC Staining of Paraffin-embedded Tissue Sections.

Western Blot Shows Human Dystroglycan Specificity by Using Knockout Cell Line. Western blot shows lysates of HEK293T human embryonic kidney parental cell line and Dystroglycan knockout HEK293T cell line (KO). PVDF membrane was probed with 1 µg/mL of Sheep Anti-Human Dystroglycan Antigen Affinity-purified Polyclonal Antibody (Catalog # AF6868) followed by HRP-conjugated Anti-Sheep IgG Secondary Antibody (Catalog # HAF016). Specific bands were detected for a--Dystroglycan at approximately 110 kDa -and beta -Dystroglycan at approximately 42 kDa (as indicated) in the parental HEK293T cell line, but is not detectable in knockout HEK293T cell line. GAPDH (Catalog # AF5718) is shown as a loading control. This experiment was conducted under reducing conditions and using Immunoblot Buffer Group 1.

Detection of Human Dystroglycan by Western Blot Hit compound 4BPPNit validation in the FKRP dystroglycanopathy human iPSC modelsFKRP‐NSCs treated with 4BPPNit showed increased IIH6 reactivity, compared with untreated control cells. Note that the augmented IIH6 reactivity by 4BPPNit is much weaker than that in CRISPR‐corrected‐NSCs. Under the same exposure time, the signal intensity of IIH6 reactivity is saturated in corrected‐NSCs.Representative immunoblots with the IIH6 antibody using FKRP‐ and corrected‐NSCs treated with 4BPPNit for 24, 48, and 72 h, and DMSO only controls.Quantification of alpha ‐dystroglycan glycosylation (IIH6), compared with DMSO only controls.Representative immunoblots of laminin‐binding analysis using FKRP‐ and corrected‐NSCs treated with 4BPPNit and DMSO only controls.Quantification of laminin‐binding activities, compared with DMSO only controls.Representative immunoblots with the AF6868 antibody detecting both alpha ‐dystroglycan core protein and beta ‐dystroglycan in FKRP‐ and corrected‐NSCs treated with 4BPPNit and DMSO only controls.Quantification of alpha ‐dystroglycan core protein and beta ‐dystroglycan expression, compared with DMSO only controls.Data information: Intensities of glycosylation, laminin‐binding activity, or protein expression are normalized to GAPDH protein expression. Note that the left and right panels of the immunoblots in (B) and (D) are not equivalent exposure time. See also Appendix Fig S5. Values indicate mean ± s.d. (n = 3 or 4 biological replicates; one‐way ANOVA; NS, not significant; *P < 0.05; **P < 0.01). Image collected and cropped by CiteAb from the following publication (https://pubmed.ncbi.nlm.nih.gov/31566294), licensed under a CC-BY license. Not internally tested by R&D Systems.