Human/Mouse ADAM9 Ectodomain Antibody

Detection of Human and Mouse ADAM9 by Western Blot. Western blot shows lysates of C2C12 mouse myoblast cell line and WI-38 human lung fibroblast cell line. PVDF membrane was probed with 1 µg/mL of Goat Anti-Human/Mouse ADAM9 Ectodomain Antigen Affinity-purified Polyclonal Antibody (Catalog # AF949) followed by HRP-conjugated Anti-Goat IgG Secondary Antibody (HAF017). Specific bands were detected for ADAM9 at approximately 110 and 80 kDa (as indicated). This experiment was conducted under reducing conditions and using Immunoblot Buffer Group 1.

Western Blot Shows Human ADAM9 Specificity by Using Knockout Cell Line. Western blot shows lysates of HeLa human cervical epithelial carcinoma parental cell line and ADAM9 knockout HeLa cell line (KO). PVDF membrane was probed with 1 µg/mL of Goat Anti-Human/Mouse ADAM9 Ectodomain Antigen Affinity-purified Polyclonal Antibody (Catalog # AF949) followed by HRP-conjugated Anti-Goat IgG Secondary Antibody (HAF017). Specific bands were detected for ADAM9 at approximately 78 and 110 kDa (as indicated) in the parental HeLa cell line, but is not detectable in knockout HeLa cell line. GAPDH (AF5718) is shown as a loading control. This experiment was conducted under reducing conditions and using Immunoblot Buffer Group 1.

Detection of Mouse ADAM9 by Western Blot Rescue of ADAM9 expression restores EMCV replication in ADAM9 KO cells. WT and ADAM9 KO HeLa cells were transduced with retroviral vectors with wild-type (WT) murine ADAM9 (mADAM9), catalytically inactive mutant ADAM9 (E>A), cytoplasmic-tail-deleted ( delta CT) ADAM9 constructs, or GFP control vectors. (A) WT, KO, and rescue cell lysates were analyzed by Western blot using two different ADAM9 antibodies. Top panel, rabbit anti-human ADAM9 that detects an epitope in the intracellular domain of human ADAM9 and cross-reacts with mouse ADAM9 (black asterisk). Middle panel, goat anti-mouse ADAM9 that detects the extracellular domain of murine ADAM9 but not human ADAM9 (blue asterisk). Bottom panel, anti-actin which detects both human and murine beta -actin. Top panel, WT but not KO cells expressed human ADAM9. Middle panel, rescue but not KO cells expressed murine ADAM9. Bottom panel, actin loading control. (B) WT clones, ADAM9 KO clones, and rescued ADAM9-expressing clones were infected with EMCV or CVB3 at various MOIs and incubated at 37°C for 24 h. Viability of EMCV-infected and CVB3-infected clones was measured by CellGlo ATP luminescence. (C) EMCV replication was quantified in infected culture supernatants by plaque assay. Neither the functional sequence of the ADAM9 metalloproteinase domain nor the cytoplasmic tail is required for EMCV infection. ***, P < 0.0001, KO versus WT and KO versus rescue. Image collected and cropped by CiteAb from the following open publication (https://pubmed.ncbi.nlm.nih.gov/30723129), licensed under a CC-BY license. Not internally tested by R&D Systems.

Detection of ADAM9 in Mouse Kidney. ADAM9 was detected in immersion fixed paraffin-embedded sections of mouse kidney using Goat Anti-Human/Mouse ADAM9 Ectodomain Antigen Affinity-purified Polyclonal Antibody (Catalog # AF949) at 10 µg/ml for 1 hour at room temperature followed by incubation with the Anti-Goat IgG VisUCyte™ HRP Polymer Antibody (Catalog # VC004). Before incubation with the primary antibody, tissue was subjected to heat-induced epitope retrieval using VisUCyte Antigen Retrieval Reagent-Basic (Catalog # VCTS021). Tissue was stained using DAB (brown) and counterstained with hematoxylin (blue). Specific staining was localized to the membrane. View our protocol for Chromogenic IHC Staining of Paraffin-embedded Tissue Sections.

Detection of ADAM9 in C2C12 cells by Flow Cytometry C2C12 cells were stained with Goat Anti-Human/Mouse ADAM9 Ectodomain Antigen Affinity-purified Polyclonal Antibody (Catalog # AF949, filled histogram) or isotype control antibody (Catalog # AB-108-C, open histogram) followed by Allophycocyanin-conjugated Anti-Goat IgG Secondary Antibody (Catalog # F0108). View our protocol for Staining Membrane-associated Proteins.