fasl antibody  (R&D Systems)

Journal: Cancer Science

Article Title: Leucine‐rich repeat neuronal protein‐1 suppresses apoptosis of gastric cancer cells through regulation of Fas/FasL

doi: 10.1111/cas.14042

Figure Lengend Snippet: Knocking down leucine‐rich repeat neuronal protein‐1 (LRRN1) increases apoptosis by activating Fas/FasL. A, Western blot analysis shows that knocked down LRRN1 increased gastric cancer (GC) apoptosis by activating Fas/FasL. B, ELISA shows that FasL level increased in cell culture medium. C, Flow cytometry shows that adding FasL antibodies can reverse the apoptosis caused by knockdown of LRRN1. D,E, Flow cytometry shows that knocking down Fas can reverse the apoptosis caused by knockdown of LRRN1 and its western blot analysis (E). Error bar = SEM, * P < 0.05, ** P < 0.01, indicates statistically significant difference

Article Snippet: Human LRRN1 antibody (catalog #AF4990) and FasL antibody (catalog #MAB126) were obtained from R&D Systems (Minneapolis, MN, USA).

Techniques: Western Blot, Enzyme-linked Immunosorbent Assay, Cell Culture, Flow Cytometry, Knockdown

Journal: Cancer Science

Article Title: Leucine‐rich repeat neuronal protein‐1 suppresses apoptosis of gastric cancer cells through regulation of Fas/FasL

doi: 10.1111/cas.14042

Figure Lengend Snippet: Knocking down leucine‐rich repeat neuronal protein‐1 (LRRN1) activating Fas/FasL by upregulating activator protein‐1 (AP‐1). A, Heat map and column chart of the differentially expressed genes in the apoptosis pathway. B, qRT‐PCR shows that knocking down LRRN1 upregulates c‐jun in mRNA level. C, Western blot analysis shows that knocking down LRRN1 upregulated both c‐jun and phosphorylated c‐jun. D, HGC‐27 and SNU‐216 cells were transfected with either negative control (NC)‐siRNA or JUN‐siRNA. JUN expression levels were detected by qRT‐PCR. E, Western blot analysis shows that knocking down JUN upregulates both c‐jun and phosphorylated c‐jun. F, Flow cytometry shows that knocking down c‐jun and JNKi treatment can reverse the apoptosis caused by knockdown of LRRN1. Error bar = SEM, * P < 0.05, ** P < 0.01, indicates statistically significant difference

Article Snippet: Human LRRN1 antibody (catalog #AF4990) and FasL antibody (catalog #MAB126) were obtained from R&D Systems (Minneapolis, MN, USA).

Techniques: Quantitative RT-PCR, Western Blot, Transfection, Negative Control, Expressing, Flow Cytometry, Knockdown

Journal: Cancer Science

Article Title: Leucine‐rich repeat neuronal protein‐1 suppresses apoptosis of gastric cancer cells through regulation of Fas/FasL

doi: 10.1111/cas.14042

Figure Lengend Snippet: Leucine‐rich repeat neuronal protein‐1 (LRRN1) suppresses gastric cancer (GC) cell proliferation in vivo. A, HGC‐27 cells stably transfected with either LRRN1 shRNA (shLRRN1) or control shRNA (shNC) were s.c. injected into athymic nude mice (n = 5 per group). B, Tumor volume was evaluated every 3 d for 27 d. C, At day 27, mice were killed, and tumors were photographed and weighed. D, TUNEL staining of the paraffin sections of the xenograft tumors. E, Xenograft tumors with LRRN1, c‐jun, Fas and FasL was immunostaining. Error bar = SEM, * P < 0.05, ** P < 0.01, indicates statistically significant difference. Scale bar, 200 μm

Article Snippet: Human LRRN1 antibody (catalog #AF4990) and FasL antibody (catalog #MAB126) were obtained from R&D Systems (Minneapolis, MN, USA).

Techniques: In Vivo, Stable Transfection, Transfection, shRNA, Control, Injection, TUNEL Assay, Staining, Immunostaining

Journal: Stem Cell Reports

Article Title: Immunological Properties of Human Embryonic Stem Cell-Derived Retinal Pigment Epithelial Cells

doi: 10.1016/j.stemcr.2018.07.009

Figure Lengend Snippet: The Role of TGF-β, FasL, and PDL1 Signaling in RPE Immunomodulation (A) The percentage of dividing CD3 + T cells was analyzed following staining of PBMCs with CFSE, activation and co-cultured for 4 days with RPE cells in the presence or absence of inhibitors of TGF-β signaling. (B and C) The percentage of apoptotic annexin V + /PI − cells was analyzed after activation and co-cultured for 1 day with RPE cells in the presence or absence of inhibitors of FasL signaling (B) or anti-PDL1 blocking antibody (α-PDL) (C); na, non-activated T cells; act, cells activated with anti-CD3/anti-CD28 antibodies; SB, SB-431542; A83, A-83-01; α-TGF-β, anti-TGF-β antibody; DcR, recombinant human DcR3; α-FasL, anti-FasL antibody. Data are presented as means ± SEM of at least 3 independent experiments. ∗ p ≤ 0.05; ∗∗ p ≤ 0.01, ∗∗∗ p ≤ 0.001.

Article Snippet: The following inhibitors were used: neutralizing anti-PDL1 Ab (2–12 μg/mL, no. AF156), neutralizing anti-FasL Ab (10 ng/mL, no. MAB126), recombinant human DcR3/TNFRSF6B chimeric protein acting as an inhibitor of FasL signaling (0.05–0.2 μg/mL, no. 142-DC-100), neutralizing anti-TGF-β1,2,3 Ab (10 μg/mL, no. MAB1835; all the inhibitors from R&D Systems, Minneapolis, MN), and two small molecules inhibiting TGF-β signaling, A-83-01 (0.1–0.5 μM) and SB-431542 (10 or 20 μM; both from Tocris Bioscience, Bristol, UK).

Techniques: Staining, Activation Assay, Cell Culture, Blocking Assay, Recombinant

Journal: Cell death & disease

Article Title: Biliary tract instillation of a SMAC mimetic induces TRAIL-dependent acute sclerosing cholangitis-like injury in mice.

doi: 10.1038/cddis.2016.459

Figure Lengend Snippet: Figure 2 Normal human cholangiocytes are moderately sensitive to SM-induced, ripoptosome-mediated apoptosis. (a) Apoptosis assessed morphologically after DAPI staining (top panels) and by caspase 3/7 activation (bottom panels) in human cholangiocyte cell lines H69 and NHC, and the human breast cancer cell line MDA-MB-231, incubated for 24 h with or without (cnt) the SMAC mimetic TL32711 (1 μM), in the presence or absence of neutralizing antibodies against TNFα (1 μg/ml) or FasL (1 μg/ml), or recombinant TRAIL-R2:Fc (1 μg/ml). (b) TNFα and TRAIL gene expression analyzed by qPCR in H69, NHC and MDA-MB-231 cells incubated in the presence of TL32711 for the indicated times. Expression normalized to GAPDH RNA. Data are expressed as fold increase over control. Mean ± s.e. are depicted from three independent experiments in H69 and NHC (c) H69 cells were treated with TL32711 and subjected to caspase 8 immunoprecipitation at the indicated time points. Affinity-purified proteins and total cell lysates (input) were analyzed by immunoblot for caspase 8, FADD and RIP1. (d) NHC cells were incubated with or without the TL32711 for 24 h, in the presence or absence of the pan- caspase inhibitor Q-VD-OPh (QVD, 10 μM) or the RIP1 kinase inhibitor necrostatin 1 (nec-1, 20 μM). Apoptosis was assessed morphologically after DAPI staining (left panel) and by caspase 3/7 activation (right panel). (e) H69 and RIP1−/−H69 (clones 1 and 2) were incubated for 24 h in the presence or absence of TL32711. Apoptosis was assessed by DAPI staining (left panel) and by caspase 3/7 activation (right panel). In (a, d and e), mean ± S.E. are depicted from three independent experiments performed in triplicate. *Po0.05, **Po0.005

Article Snippet: The human anti-TNFα neutralizing antibody (AF210) and human anti-FasL neutralizing antibody (MAB126) were from R&D Systems (Minneapolis, MN, USA).

Techniques: Staining, Activation Assay, Incubation, Recombinant, Gene Expression, Expressing, Control, Immunoprecipitation, Affinity Purification, Western Blot, Clone Assay