antibody rela clone c-20  (Santa Cruz Biotechnology)

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: IκBα nuclear export enables 4–1BB induced cRel activation and IL-2 production to promote CD8 T cell immunity

doi: 10.4049/jimmunol.2000039

Figure Lengend Snippet: (A) Naive NfkbiaWT/WT (WT) and NfkbiaNES/NES (NES) T cells were stimulated with 5 μg/mL each anti-CD3 and anti-CD28 antibodies and cell lysates were prepared. Supershift EMSA analysis with anti-cRel and anti-ReA antibodies was used to detect RelA and cRel complexes, respectively. Below, quantification of RelA (left, *) and cRel (right, **) binding, relative to unstimulated WT T cells and normalized to Oct-1 binding, is shown as fold change in comparison to unstimulated samples from three independent experiments with the mean +/− SEM with statistical significance at *p < 0.05 or as indicated.

Article Snippet: For supershift assays, 1 μL of each antibody (0.2 μg) for RelA (Santa Cruz, clone C-20), cRel (Santa Cruz, clone C) or RelB (Santa Cruz, clone C-19) was added to cell extracts, with the reaction volume not exceeding 10 μL.

Techniques: Binding Assay, Comparison

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: IκBα nuclear export enables 4–1BB induced cRel activation and IL-2 production to promote CD8 T cell immunity

doi: 10.4049/jimmunol.2000039

Figure Lengend Snippet: (A) Naïve WT and NfkbiaNES/NES CD8 T cells were stimulated for 24 hours with 5 μg/mL anti-CD3 and anti-CD28 antibodies with or without 0.05 μg/mL anti-4–1BB antibody. Supershift analysis using anti-cRel and anti-RelA antibodies was performed to detect RelA (left, *) and cRel (right, **) complexes, respectively. Below, quantification of indicated complexes is shown as fold change in comparison to unstimulated samples and normalized to Oct-1 binding. Data plotted is the mean +/− SEM and includes 3 independent experiments with statistical significance at *p < 0.05.

Article Snippet: For supershift assays, 1 μL of each antibody (0.2 μg) for RelA (Santa Cruz, clone C-20), cRel (Santa Cruz, clone C) or RelB (Santa Cruz, clone C-19) was added to cell extracts, with the reaction volume not exceeding 10 μL.

Techniques: Comparison, Binding Assay

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: IκBα nuclear export enables 4–1BB induced cRel activation and IL-2 production to promote CD8 T cell immunity

doi: 10.4049/jimmunol.2000039

Figure Lengend Snippet: (A) Naive WT and NfkbiaNES/NES CD8 T cells were fixed, permeabilized, and stained with anti-IκBα, RelA or cRel antibodies and the DRAQ5 nuclear dye. WT cells were treated with 20 ng/ml of Leptomycin B for 45 minutes as a control. Single cell image analysis by ImageStream flow cytometry. Representative images from each group are shown at an equivalent magnification (above) and the similarity score for each antibody staining is plotted. Data plotted includes 3 independent experiments (see Methods).

Article Snippet: For supershift assays, 1 μL of each antibody (0.2 μg) for RelA (Santa Cruz, clone C-20), cRel (Santa Cruz, clone C) or RelB (Santa Cruz, clone C-19) was added to cell extracts, with the reaction volume not exceeding 10 μL.

Techniques: Staining, Control, Flow Cytometry

Journal: Immunity

Article Title: NEMO Prevents RIP Kinase 1-Mediated Epithelial Cell Death and Chronic Intestinal Inflammation by NF-κB-Dependent and -Independent Functions

doi: 10.1016/j.immuni.2016.02.020

Figure Lengend Snippet: Combined Deficiency in FADD and RIPK3 Prevents IEC Death, Paneth Cell Loss, and Colitis in NEMO IEC-KO Mice (A and F) Representative images of colon and ileal sections from mice with the indicated genotypes. (B) Immunoblot analysis of protein extracts from primary small intestinal IECs from mice with the indicated genotypes and with the indicated antibodies. (C and G) Graphs depicting histopathological scores of colon and ileal sections from mice with the indicated genotypes (n = 5 or 6 mice per genotype). (D and I) Graphs depicting the percentage of crypts with cl. casp. 3 stained cells on colon and ileal sections from littermates with the indicated genotypes (n = 5 or 6 mice per genotype). (E, J, and K) Graphs depicting mRNA levels of the indicated genes in the colon and ileum of mice with the indicated genotypes (n = 9 or 10 mice per genotype). (H) Graph depicting Paneth cell scores of ileal sections from mice with the indicated genotypes (n = 5 or 6 mice per genotype). Scale bars represent 100 μm.

Article Snippet: Primary antibodies used for immunoblot analysis: RelA C-20 (sc-372), RelB C-19 (sc-226), and c-Rel (sc-71), β-actin 1-19 (sc-161) and HDAC1 H-51 (sc-7872) from Santa Cruz, TNFR1 (D317K); Caspase 3 (9662), cleaved Caspase 3 (9661) from Cell Signaling; RIPK1 (610459) from BD; RIPK3 (ADI-905-242-100) from Enzo Life Sciences; FADD (1F7) 05-486 from Upstate; Cre 69050-3 from Novagen; Caspase-8 (ALX-804-447) from Alexis; and NEMO (homemade rabbit polyclonal serum) and α-tubulin (T6074) from Sigma.

Techniques: Western Blot, Staining

Journal: Immunity

Article Title: NEMO Prevents RIP Kinase 1-Mediated Epithelial Cell Death and Chronic Intestinal Inflammation by NF-κB-Dependent and -Independent Functions

doi: 10.1016/j.immuni.2016.02.020

Figure Lengend Snippet: Lack of RIPK1 Kinase Activity Prevents Colitis Development and Paneth Cell Loss in NEMO IEC-KO Mice (A and E) Representative images of colon and ileal sections from mice with the indicated genotypes. (B and F) Graphs depicting histopathological scores of colon and ileal sections from mice with the indicated genotypes (n = 6–13 mice per genotype). (C and I) Graphs depicting the percentage of crypts with cl. casp. 3 stained cells on colon and ileal sections from littermates with the indicated genotypes (n = 4–12 mice per genotype). (D, H) Graphs depicting mRNA levels of the indicated genes in the colon and ileum of mice with the indicated genotypes (n = 6 or 7 mice per genotype). (G) Graph depicting Paneth cell scores of ileal sections from mice with the indicated genotypes (n = 6–10 mice per genotype). (J) Immunoblot analysis of the indicated proteins co-immunoprecipitated with FADD from total colon lysates from mice with the indicated genotypes (one out of three independent experiments shown). Scale bars represent 100 μm.

Article Snippet: Primary antibodies used for immunoblot analysis: RelA C-20 (sc-372), RelB C-19 (sc-226), and c-Rel (sc-71), β-actin 1-19 (sc-161) and HDAC1 H-51 (sc-7872) from Santa Cruz, TNFR1 (D317K); Caspase 3 (9662), cleaved Caspase 3 (9661) from Cell Signaling; RIPK1 (610459) from BD; RIPK3 (ADI-905-242-100) from Enzo Life Sciences; FADD (1F7) 05-486 from Upstate; Cre 69050-3 from Novagen; Caspase-8 (ALX-804-447) from Alexis; and NEMO (homemade rabbit polyclonal serum) and α-tubulin (T6074) from Sigma.

Techniques: Activity Assay, Staining, Western Blot, Immunoprecipitation

Journal: Molecular Cancer

Article Title: PKM2 promotes tumor angiogenesis by regulating HIF-1α through NF-κB activation

doi: 10.1186/s12943-015-0490-2

Figure Lengend Snippet: PKM2 mediates hypoxia-triggered VEGF-A secretion by activation of NF-κB/p65 subunit. a immunoprecipitation of endogenous p65 was performed with lysates of PaTu2 and BxPC3 cells. Membranes were incubated with PKM2 and reprobed with p65 antibodies. b , c PaTu2 and Capan1 cancer cell lines with abrogated p65/Rel (shp65 #G3 or shp65 #F12) were transiently transfected with VEGF-A-luc and pTK-Renilla. Four hours later cells were subjected to normoxic or hypoxic conditions. After 24 h cell lysates were prepared and subjected to luciferase assay. Bars are the means +/- SEM of at least three independent experiments. d supernatants of PaTu2 or Capan1 cells transduced with p65/RelA-specific shRNA (shp65) or a non-coding shRNA incubated in low oxygen were subjected to VEGF-A-specific ELISA. Bars are the means +/- SEM of at least two independent experiments conducted in duplicate. e PaTu2 and Capan1 cancer cell lines with abrogated PKM2 were transiently transfected with 3xκB-luc and pTK-Renilla. After incubation under normoxic or hypoxic conditions cell lysates were prepared and reporter activity assayed. Bars are the means +/- SEM of at least three independent experiments. f , g pancreatic Capan1 cells with abrogated PKM2 were transiently co-transfected with p65 and VEGF-A-luc reporter before incubation in low oxygen atmosphere. Luciferase was measured after 24 h. h supernatants of Capan1 cells with deleted PKM2 and overexpressing p65 cultivated in O 2 -deprived atmosphere or normoxia were subjected to VEGF-A-specific ELISA. Bars are the means +/- SEM of at least two independent experiments conducted in duplicate (No – normoxia; Hy – hypoxia)

Article Snippet: The following antibodies were used: PKM2 (Abcam, #ab55602); cleaved PARP (Cell Signaling, #9542S); HIF-1α (BD Transduction Laboratories, #610959); p65/RelA C-20 (Santa Cruz, #sc-372), GFP (Roche, # 1052 1400) and β-actin (Sigma, #A1978).

Techniques: Activation Assay, Immunoprecipitation, Incubation, Transfection, Luciferase, Transduction, shRNA, Enzyme-linked Immunosorbent Assay, Activity Assay

Journal: Molecular Cancer

Article Title: PKM2 promotes tumor angiogenesis by regulating HIF-1α through NF-κB activation

doi: 10.1186/s12943-015-0490-2

Figure Lengend Snippet: PKM2 regulates HIF-1α transcription via activation of NF-κB signaling pathway. a pancreatic cancer cells were transiently transfected with a p65 expression plasmid (YFP-p65). In parallel, cells transfected with control empty vector (GFP-empty) were cultivated in normoxia or hypoxia for 8 h. Lysates were subjected to SDS-PAGE and membranes were incubated with HIF-1α and p65 antibodies. β-actin was used as loading control. b PaTu2 cells were co-transfected with p65 and HIF-1α reporter. After incubation for 24 h in low oxygen atmosphere, lysates were prepared and luciferase was measured. c cancer cells with abrogated p65 were transfected 3xHRE-luc and then incubated in low oxygen atmosphere. Luciferase was measured 28 h after transfection. Bars are the means +/- SEM of at least three independent experiments. d PaTu2 cells featuring p65 knock-down were co-transfected with PKM2 expression plasmid and 3xHRE-luc and then grown in an atmosphere deprived of oxygen. Luciferase was scored 24 h later. Bars are the means +/- SEM of at least three independent experiments. e PaTu2 and Capan1 cancer cells with depleted PKM2 were co-transfected with a p65 expression plasmid and 3xHRE-luc and then grown in an atmosphere deprived of oxygen. Luciferase was scored 24 h later. Bars are the means +/- SEM of at least three independent experiments. f PaTu2 cells with abrogated PKM2 were co-transfected with a HIF-1α expression plasmid and a 3xκB-luc reporter and then grown in hypoxia. Luciferase was scored 24 h later. Bars are the means +/- SEM of at least three independent experiments. For all reporter assays TK-Renilla was used as internal control

Article Snippet: The following antibodies were used: PKM2 (Abcam, #ab55602); cleaved PARP (Cell Signaling, #9542S); HIF-1α (BD Transduction Laboratories, #610959); p65/RelA C-20 (Santa Cruz, #sc-372), GFP (Roche, # 1052 1400) and β-actin (Sigma, #A1978).

Techniques: Activation Assay, Transfection, Expressing, Plasmid Preparation, Control, SDS Page, Incubation, Luciferase, Knockdown

Journal: Molecular Cancer

Article Title: PKM2 promotes tumor angiogenesis by regulating HIF-1α through NF-κB activation

doi: 10.1186/s12943-015-0490-2

Figure Lengend Snippet: PKM2 contributes to tumor growth and angiogenesis by regulating HIF-1α via NF-κB activation. a in physiological conditions PKM2 and p65 reside in the cytosol. Hypoxic insult is followed by translocation of PKM2 and p65 to the nucleus in pancreatic cancer cells. Following the interaction with PKM2, NF-κB subunit p65 activates the transcription of HIF-1α and its target gene VEGF-A. As a result, augmented secretion of VEGF translates to a boost in blood vessel formation, which in turn contributes to tumor growth. b in the absence of PKM2, transcription and secretion of VEGF-A is still present, yet to a lower level. While our data do not exclude the VEGF secretion as a result of sole NF-κB activation or HIF-1α accumulation, they emphasize the essential role of PKM2 in the regulation of HIF-1α and NF-κB transcription factor during tumor angiogenesis and tumor growth of hypoxic pancreatic tumors

Article Snippet: The following antibodies were used: PKM2 (Abcam, #ab55602); cleaved PARP (Cell Signaling, #9542S); HIF-1α (BD Transduction Laboratories, #610959); p65/RelA C-20 (Santa Cruz, #sc-372), GFP (Roche, # 1052 1400) and β-actin (Sigma, #A1978).

Techniques: Activation Assay, Translocation Assay