Human cIAP-1/HIAP-2 Antibody

Detection of Human cIAP‑1/HIAP‑2 by Western Blot. Western blot shows lysates of HEK293 human embryonic kidney cell line transfected with human cIAP-1 (lane 1), human cIAP-2 (lane 2), or non-transfected (lane 3). PVDF membrane was probed with 0.5 µg/mL Goat Anti-Human cIAP-1/HIAP-2 Antigen Affinity-purified Polyclonal Antibody (Catalog # AF8181) followed by HRP-conjugated Anti-Goat IgG Secondary Antibody (Catalog # HAF017). For additional reference, lystates of HepG2 human hepatocellular carcinoma cell line and Jurkat human acute T cell leukemia cell line were included. A specific band for cIAP-1/HIAP-2 was detected at approximately 65 kDa (as indicated). This experiment was conducted under reducing conditions and using Immunoblot Buffer Group 2.

cIAP‑1/HIAP‑2 in Human Lymph Node. cIAP-1/HIAP-2 was detected in immersion fixed paraffin-embedded sections of human lymph node using Goat Anti-Human cIAP-1/HIAP-2 Antigen Affinity-purified Polyclonal Antibody (Catalog # AF8181) at 10 µg/mL overnight at 4 °C. Tissue was stained using the Anti-Goat HRP-DAB Cell & Tissue Staining Kit (brown; Catalog # CTS008) and counterstained with hematoxylin (blue). Specific staining was localized to lymphocytes. View our protocol for Chromogenic IHC Staining of Paraffin-embedded Tissue Sections.

cIAP‑1/HIAP‑2 in Human Lymphoma. cIAP-1/HIAP-2 was detected in immersion fixed paraffin-embedded sections of human lymphoma using Goat Anti-Human cIAP-1/HIAP-2 Antigen Affinity-purified Polyclonal Antibody (Catalog # AF8181) at 15 µg/mL overnight at 4 °C. Tissue was stained using the Anti-Goat HRP-DAB Cell & Tissue Staining Kit (brown; Catalog # CTS008) and counterstained with hematoxylin (blue). Lower panel shows a lack of labeling if primary antibodies are omitted and tissue is stained only with secondary antibody followed by incubation with detection reagents. View our protocol for Chromogenic IHC Staining of Paraffin-embedded Tissue Sections.

Detection of Human cIAP‑1/HIAP‑2 by Simple Western^TM. Simple Western lane view shows lysates of HepG2 human hepatocellular carcinoma cell line, loaded at 0.5 mg/mL. A specific band was detected for cIAP-1/HIAP-2 at approximately 66 kDa (as indicated) using 5 µg/mL of Goat Anti-Human cIAP-1/HIAP-2 Antigen Affinity-purified Polyclonal Antibody (Catalog # AF8181) followed by 1:50 dilution of HRP-conjugated Anti-Goat IgG Secondary Antibody (Catalog # HAF019). This experiment was conducted under reducing conditions and using the 12-230 kDa separation system.

Western Blot Shows Human cIAP‑1/HIAP‑2 Specificity by Using Knockout Cell Line. Western blot shows lysates of HeLa human cervical epithelial carcinoma parental cell line and cIAP-1/HIAP-2 knockout HeLa cell line (KO). PVDF membrane was probed with 0.5 µg/mL of Goat Anti-Human cIAP-1/HIAP-2 Antigen Affinity-purified Polyclonal Antibody (Catalog # AF8181) followed by HRP-conjugated Anti-Goat IgG Secondary Antibody (Catalog # HAF017). A specific band was detected for cIAP-1/HIAP-2 at approximately 68 kDa (as indicated) in the parental HeLa cell line, but is not detectable in knockout HeLa cell line. GAPDH (Catalog # AF5718) is shown as a loading control. This experiment was conducted under reducing conditions and using Immunoblot Buffer Group 1.

Specificity of Human cIAP‑1/HIAP‑2 by Simple Western^TM. Simple Western lane view shows lysates of HeLa human cervical epithelial carcinoma parental cell line and cIAP-1/HIAP-2 knockout HeLa cell line (KO), loaded at 0.2 mg/mL. A specific band was detected for cIAP-1/HIAP-2 at approximately 66 kDa (as indicated) in the parental HeLa cell line, but is not detectable in knockout HeLa cell line. Goat Anti-Human cIAP-1/HIAP-2 Antigen Affinity-purified Polyclonal Antibody (Catalog # AF8181) was used at 5 µg/mL followed by 1:50 dilution of HRP-conjugated Anti-Goat IgG Secondary Antibody (Catalog # HAF019). This experiment was conducted under reducing conditions and using the 12-230 kDa separation system.

Detection of Human cIAP-1/HIAP-2 by Western Blot IAP expression in glioblastoma cell lines. Expression levels of cIAP1, cIAP2, XIAP and ML-IAP were analyzed by western blotting and quantified in U87MG and GL261 adherent GBM cell lines, and in GBM6 and GBM9 spheres. Expression level of beta -actin served as loading control. The four GBM cell lines expressed heterogeneously cIAP1, cIAP2, XIAP and ML-IAP. A representative experiment of four experiments is shown. Quantification was performed using ImageJ software (National Institutes of Health, Bethesda, MD, USA) and data presented were normalized to beta -actin expression Image collected and cropped by CiteAb from the following publication (https://pubmed.ncbi.nlm.nih.gov/27490930), licensed under a CC-BY license. Not internally tested by R&D Systems.

Detection of Human cIAP-1/HIAP-2 by Western Blot cIAP-1 and cIAP-2 are downregulated in cholangiocytes of PSC patients. (a) Representative images of liver sections stained for cIAP-1 (left panel) and cIAP-2 (right panel) in patients with normal, NASH and PSC (stage IV) liver histology. Photomicrographs of small bile ducts (SBD) and large bile ducts (LBD) taken at × 20 magnification. The arrows point to the bile ducts. (b) Histological scoring for cIAP-1 in normal (5, 31), NASH (12, 120) and PSC (16, 131) patients and for cIAP-2 in normal (5, 31), NASH (12, 118) and PSC (16, 123) patients. Numbers in parentheses indicate total number of patients and total number of small and large bile ducts evaluated, respectively. Grade 0=no protein expression; grade 3=high protein expression. **P<0.01, ***P<0.001. (c) Representative images of liver sections stained for TWEAK (left panel) and Fn14 (right panel) from patients with normal or PSC (stage IV) liver histology. Photomicrographs of small bile ducts and large bile ducts taken at × 40 and × 20 magnification, respectively. The arrows point to the bile ducts. (d) Immunoblot analysis showing expression of cIAP-1, cIAP-2 and actin (loading control) in H69 cells treated with human recombinant TWEAK (100 ng/ml) for the indicated times Image collected and cropped by CiteAb from the following publication (https://www.nature.com/articles/cddis2016459), licensed under a CC-BY license. Not internally tested by R&D Systems.

Detection of Human cIAP-1/HIAP-2 by Immunocytochemistry/Immunofluorescence Prognostic value of cIAP1, cIAP2, XIAP and ML-IAP protein expression in human glioblastomas (cohorts 1 and 2). (a) cIAP1-, cIAP2-, XIAP- and ML-IAP-positive stainings in GBM. IAPs were heterogeneously expressed by tumor cells in GBM samples (Table 1). Stainings were diffused with a stronger punctuated positivity into the cytoplasm. Black arrows highlight cIAP2-positive nuclei. Scale bars, 50 μm. (b) Correlation of ML-IAP protein expression with PFS and OS in cohort 1. The cutoff was 35% and was determined by performing a ROC curve. ML-IAP expression of ⩾35% was correlated with a poor prognosis. (c) Correlation of ML-IAP protein expression with PFS and OS in cohort 2. The cutoff was the same as that for cohort 1 analysis (35%). ML-IAP expression of ⩾35% was correlated with a poor prognosis Image collected and cropped by CiteAb from the following publication (https://pubmed.ncbi.nlm.nih.gov/27490930), licensed under a CC-BY license. Not internally tested by R&D Systems.

Detection of Human cIAP-1/HIAP-2 by Western Blot Apoptosis and IAP expression upon SMAC mimetic GDC-0152 treatment in glioblastoma cell lines. (a) Apoptosis (SubG0/G1) of DMSO control and GDC-0152-treated cells was determined by flow cytometry of propidium iodide-stained nuclei and percentage of apoptosis is shown. U87MG and GL261 cell lines were treated for 72 h and GBM6 and GBM9 cell lines were treated for 8 days at the indicated concentrations. At these respective time points, percentage of U87MG cells dead by apoptosis, percentage of GL261 cells, percentage of GBM6 cells and percentage of GBM9 cells. Data are expressed as mean+S.E.M. Three independent experiments were performed for the GL261 cell lines and five for the U87MG, GBM6 and GBM9 cell lines. *P<0.05; **P<0.01; ***P<0.005. (b) Expression levels of cIAP1, cIAP2, XIAP and ML-IAP were analyzed by western blotting. Cell lines were treated with 1 μM of GDC-0152. U87MG and GL261 were treated for 72 h and GBM6 and GBM9 cell lines for 8 days. In all GBM cell lines GDC-0152 decreased IAP expression. Expression level of beta -actin served as loading control. A representative experiment of three experiments is shown Image collected and cropped by CiteAb from the following publication (https://pubmed.ncbi.nlm.nih.gov/27490930), licensed under a CC-BY license. Not internally tested by R&D Systems.