Western Blot:Article Title: Rtp801 , a suppressor of mTOR signaling, is an essential mediator of cigarette smoke – induced pulmonary injury and emphysema
Article Snippet: .. NORMAL SMOKER COPD G2 COPD G3 COPD G4 Total 12 13 8 8 16 Males/Females 10/2 8/5 5/8 5/8 10/16 Age 37 [24.8-49.3] 59 [54.4-63.6] 59.7 [54.7-64.7] 58.8 [54,5-63] 56.5 [54-58.9] FEV1/FVC NA 0.76 [0.72-0.80] 0.56 [0.50-0.62] 0.41 [0.25-0.46] 0.26 [0.21-0.30] Pack Years - 38 [24-53] 40 [5-75] 55 [30-79] 59 [38-80] Smoking-free years - 12 [6-17] 6 [0-18] 5 [0-14] 5 1-8] Supplementary Methods Antibodies and reagents Antibodies against: RTP801/REDD1: rabbit anti – human RTP801, rabbit anti – mouse RTP801 antibodies (Quark Pharmaceuticals Inc), and rabbit anti – RTP801 (Santa Cruz Biotech.) used for immunoblotting; chicken – anti – human antibodies (Chemicon International), used for IHC analysis; goat anti – RTP801/REDD1 (Santa Cruz Biotech, sc – 46034) was used for colocalization immunofluorescence; activated caspase 3, cleaved poly – ADP ribose polymerase (PARP) (Asp214) (7C9) and p38 MAP Kinase, phospho – p38 MAP Kinase (Thr180/Tyr182), phospho – NF – B p65 (Ser536 and Ser276), NF – B p65 (for Western blots), phospho – S6 ribosomal protein (Ser235/236), and S6 ribosomal protein (Cell Signaling Technology); polyclonal NF – κB p65 (sc – 46034; Santa Cruz Biotech., for IHC); IκBα (Cell Signaling); anti – macrophage Mac – 3 (BD Bioscience), rat anti – neutrophil directed against a polymorphic 40kD antigen (Serotec), active caspase 3 and F4/80 (Abcam), prosurfactant protein C (proSP – C) (Chemicon International, AB3786), T1α (hamster) (generous gift by Dr. Mary Williams); thrombomodulin (R&D Systems, AF3894); 3 – nitrotyrosine (Cayman Chemical); 8 oxo – 2’deoxy – guanosine (QED) ; Assessment of oxidative stress: 2’,7’ – dichlorofluorescin diacetate (DCF – DA) (Molecular Probe). .. Plasmids, transfection reagents, and detection systems: pcDNA3.1(+) (Invitrogen Corporation); pHTS NF – B reporter vector (Biomyx Tecgnology), pRL – TK vector encoding Renilla luciferase (Promega), Superfect (Qiagen), Lipofectamine 2000 (Invitrogen), jet PEI (Qbio gene) and Dual Stop and Grow luciferase assay system (Promega), RTP801 promoter constructs.
Immunohistochemistry:Article Title: Rtp801 , a suppressor of mTOR signaling, is an essential mediator of cigarette smoke – induced pulmonary injury and emphysema
Article Snippet: .. NORMAL SMOKER COPD G2 COPD G3 COPD G4 Total 12 13 8 8 16 Males/Females 10/2 8/5 5/8 5/8 10/16 Age 37 [24.8-49.3] 59 [54.4-63.6] 59.7 [54.7-64.7] 58.8 [54,5-63] 56.5 [54-58.9] FEV1/FVC NA 0.76 [0.72-0.80] 0.56 [0.50-0.62] 0.41 [0.25-0.46] 0.26 [0.21-0.30] Pack Years - 38 [24-53] 40 [5-75] 55 [30-79] 59 [38-80] Smoking-free years - 12 [6-17] 6 [0-18] 5 [0-14] 5 1-8] Supplementary Methods Antibodies and reagents Antibodies against: RTP801/REDD1: rabbit anti – human RTP801, rabbit anti – mouse RTP801 antibodies (Quark Pharmaceuticals Inc), and rabbit anti – RTP801 (Santa Cruz Biotech.) used for immunoblotting; chicken – anti – human antibodies (Chemicon International), used for IHC analysis; goat anti – RTP801/REDD1 (Santa Cruz Biotech, sc – 46034) was used for colocalization immunofluorescence; activated caspase 3, cleaved poly – ADP ribose polymerase (PARP) (Asp214) (7C9) and p38 MAP Kinase, phospho – p38 MAP Kinase (Thr180/Tyr182), phospho – NF – B p65 (Ser536 and Ser276), NF – B p65 (for Western blots), phospho – S6 ribosomal protein (Ser235/236), and S6 ribosomal protein (Cell Signaling Technology); polyclonal NF – κB p65 (sc – 46034; Santa Cruz Biotech., for IHC); IκBα (Cell Signaling); anti – macrophage Mac – 3 (BD Bioscience), rat anti – neutrophil directed against a polymorphic 40kD antigen (Serotec), active caspase 3 and F4/80 (Abcam), prosurfactant protein C (proSP – C) (Chemicon International, AB3786), T1α (hamster) (generous gift by Dr. Mary Williams); thrombomodulin (R&D Systems, AF3894); 3 – nitrotyrosine (Cayman Chemical); 8 oxo – 2’deoxy – guanosine (QED) ; Assessment of oxidative stress: 2’,7’ – dichlorofluorescin diacetate (DCF – DA) (Molecular Probe). .. Plasmids, transfection reagents, and detection systems: pcDNA3.1(+) (Invitrogen Corporation); pHTS NF – B reporter vector (Biomyx Tecgnology), pRL – TK vector encoding Renilla luciferase (Promega), Superfect (Qiagen), Lipofectamine 2000 (Invitrogen), jet PEI (Qbio gene) and Dual Stop and Grow luciferase assay system (Promega), RTP801 promoter constructs.
Immunofluorescence:Article Title: Rtp801 , a suppressor of mTOR signaling, is an essential mediator of cigarette smoke – induced pulmonary injury and emphysema
Article Snippet: .. NORMAL SMOKER COPD G2 COPD G3 COPD G4 Total 12 13 8 8 16 Males/Females 10/2 8/5 5/8 5/8 10/16 Age 37 [24.8-49.3] 59 [54.4-63.6] 59.7 [54.7-64.7] 58.8 [54,5-63] 56.5 [54-58.9] FEV1/FVC NA 0.76 [0.72-0.80] 0.56 [0.50-0.62] 0.41 [0.25-0.46] 0.26 [0.21-0.30] Pack Years - 38 [24-53] 40 [5-75] 55 [30-79] 59 [38-80] Smoking-free years - 12 [6-17] 6 [0-18] 5 [0-14] 5 1-8] Supplementary Methods Antibodies and reagents Antibodies against: RTP801/REDD1: rabbit anti – human RTP801, rabbit anti – mouse RTP801 antibodies (Quark Pharmaceuticals Inc), and rabbit anti – RTP801 (Santa Cruz Biotech.) used for immunoblotting; chicken – anti – human antibodies (Chemicon International), used for IHC analysis; goat anti – RTP801/REDD1 (Santa Cruz Biotech, sc – 46034) was used for colocalization immunofluorescence; activated caspase 3, cleaved poly – ADP ribose polymerase (PARP) (Asp214) (7C9) and p38 MAP Kinase, phospho – p38 MAP Kinase (Thr180/Tyr182), phospho – NF – B p65 (Ser536 and Ser276), NF – B p65 (for Western blots), phospho – S6 ribosomal protein (Ser235/236), and S6 ribosomal protein (Cell Signaling Technology); polyclonal NF – κB p65 (sc – 46034; Santa Cruz Biotech., for IHC); IκBα (Cell Signaling); anti – macrophage Mac – 3 (BD Bioscience), rat anti – neutrophil directed against a polymorphic 40kD antigen (Serotec), active caspase 3 and F4/80 (Abcam), prosurfactant protein C (proSP – C) (Chemicon International, AB3786), T1α (hamster) (generous gift by Dr. Mary Williams); thrombomodulin (R&D Systems, AF3894); 3 – nitrotyrosine (Cayman Chemical); 8 oxo – 2’deoxy – guanosine (QED) ; Assessment of oxidative stress: 2’,7’ – dichlorofluorescin diacetate (DCF – DA) (Molecular Probe). .. Plasmids, transfection reagents, and detection systems: pcDNA3.1(+) (Invitrogen Corporation); pHTS NF – B reporter vector (Biomyx Tecgnology), pRL – TK vector encoding Renilla luciferase (Promega), Superfect (Qiagen), Lipofectamine 2000 (Invitrogen), jet PEI (Qbio gene) and Dual Stop and Grow luciferase assay system (Promega), RTP801 promoter constructs.
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