Journal: Nature Communications

Article Title: The cytoplasmic nuclear receptor RARγ controls RIP1 initiated cell death when cIAP activity is inhibited

doi: 10.1038/s41467-017-00496-6

Figure Lengend Snippet: RARγ C fragment are important for protecting cell from TNF-induced necroptosis via interaction with RIP1. a Immunoprecipitation of HT-29 cells treated with TSZ for the indicated times. Cell lysates were immunoprecipitated with anti-RARγ antibody and analyzed by immunoblotting with the indicated antibodies. b HEK293T cells were co-transfected with wild-type V5-RARγ (WT) or the V5-RARγ-NLSmut (NLS) and with or without FLAG-RIP1 plasmids. Cell lysates were immunoprecipitated using anti-FLAG (RIP1) antibody and analyzed with the indicated antibodies. c Scheme of RARγ and RIP1 genes ( upper panel ). HEK293T cells were co-transfected with different fragment of RARγ-V5 (F: full; N:1–201aa; C: 188–454aa) and with or without FLAG-RIP1 plasmids; or with different fragment of His-Xpress-RIP1 (F: full; N: 1–324aa; C: 324–671aa; DD: 560–669aa) and with or without RARγ-V5. Cell lysates were immunoprecipitated using anti-FLAG (RIP1) or anti-V5 (RARγ) antibody and analyzed with the indicated antibodies. d HT-29 RARγ-shRNA-A cells infected with rRARγ-C. Cell death analysis of HT-29 cont-shRNA, RARγ-shRNA-A, and RARγ-shRNA-A + rRARγ-C when treated with TSZ for 24 h was determined by PI staining using flow cytometry ( upper left panel ). (* P < 0.05 versus cont-shRNA; # P < 0.05 versus RARγ-shRNA-A; ANOVA). The bars represent the mean ± s.e.m. of three experiments. Western blot analysis of cells as mentioned in left panel ( upper right panel ). Confocal microscopy of HT-29 cells infected with GFP-RARγ-C plasmid ( lower left panel ) ( blue : DAPI; green : RARγ). Western blot analysis of HEK293T cells were transfected with p-EGFP, p-EGFP-RARγ and p-EGFP-RARγ-C by using anti-GFP and anti-Actin antibodies ( lower right panel ). All blots and images above are representative of one of three experiments

Article Snippet: Anti-RARγ (C-15) (sc-550) for human, anti-RARα (C-20) (sc-551), anti-caspase-8 (C-20) (sc-6136), anti-cIAP2 (sc7944) and anti-Fas (C-20) (sc-715) from Santa Cruz; anti-RIP1 (610459) and anti-FADD (610400) from BD Biosciences; anti-RARγ1 (ab5905) for mouse, anti-RIP3 (ab72106), anti-MLKL (ab184718) for human, anti-MLKL (ab172868) for mouse and anti-cIAP1 (ab2399) from Abcam; anti-RIP3 (2283) for mouse from ProSci, anti-TRADD (05-473) from Upstate; anti-TRAF2 (MAB3277) and anti-TNFR1 (AF-425-PB) from R&D; anti-Actin (A3853) (dilution 1:10,000), anti-FLAG (F9291) (dilution 1:5,000) and anti-GFP (G6539) (dilution 1:5 000) from Sigma; anti-V5 (R960-25) (dilution 1:5,000) from Invitrogen; anti-PARP1 (BML-SA250-0050) from Enzo Life Science; anti-GAPDH (NB300-22) (dilution 1:5,000) from Novus Biologicals; anti-cleaved caspase-8 (9496), anti-CYLD (4495), anti-RIP1 (137451) and anti-p-RIP1(65746) from Cell Signaling Technology; anti-DsRed (632392) (dilution 1:5,000) from Clontech.

Techniques: Immunoprecipitation, Western Blot, Transfection, shRNA, Infection, Staining, Flow Cytometry, Confocal Microscopy, Plasmid Preparation

Journal: Nature Communications

Article Title: The cytoplasmic nuclear receptor RARγ controls RIP1 initiated cell death when cIAP activity is inhibited

doi: 10.1038/s41467-017-00496-6

Figure Lengend Snippet: RARγ initiates the formation of death complexes by dissociating RIP1 from TNFR1. a Immunoprecipitation of HT-29 cont-shRNA, TRADD-shRNA or RIP1-shRNA treated with TSZ for the indicated times. Cell lysates were immunoprecipitated using anti-TNFR1 antibody and immunoblotted with the indicated antibodies. b Sequential immunoprecipitation of HEK293T cells co-transfected with FLAG-TNFR1, DsRed-RIP1 and increasing amounts of V5-RARγ-NLSmut plasmids as indicated. First IP : cell lysates were immunoprecipitated with anti-FLAG (TNFR1) antibody. Second IP : the remaining lysates were then immunoprecipitated with anti-V5 (RARγ) antibody. The immunoprecipitated complexes were analyzed by with the indicated antibodies. c Sequential immunoprecipitation of HEK293T cells co-transfected with V5-RARγ-NLSmut, RIP1-Myc, and increasing amounts of DsRed-RIP3 plasmids as indicated. First IP : cell lysates were immunoprecipitated with anti-V5 (RARγ) antibody. Second IP : the remaining lysates were then immunoprecipitated with anti-DsRed (RIP3) antibody. The immunoprecipitated complexes were analyzed with the indicated antibodies. d WT and RIP3−/− MEFs treated with TSZ for the indicated times. Cell lysates were immunoprecipitated using anti-Caspase-8 antibody and immunoblotted with the indicated antibodies. All blots above are representative of one of three experiments

Article Snippet: Anti-RARγ (C-15) (sc-550) for human, anti-RARα (C-20) (sc-551), anti-caspase-8 (C-20) (sc-6136), anti-cIAP2 (sc7944) and anti-Fas (C-20) (sc-715) from Santa Cruz; anti-RIP1 (610459) and anti-FADD (610400) from BD Biosciences; anti-RARγ1 (ab5905) for mouse, anti-RIP3 (ab72106), anti-MLKL (ab184718) for human, anti-MLKL (ab172868) for mouse and anti-cIAP1 (ab2399) from Abcam; anti-RIP3 (2283) for mouse from ProSci, anti-TRADD (05-473) from Upstate; anti-TRAF2 (MAB3277) and anti-TNFR1 (AF-425-PB) from R&D; anti-Actin (A3853) (dilution 1:10,000), anti-FLAG (F9291) (dilution 1:5,000) and anti-GFP (G6539) (dilution 1:5 000) from Sigma; anti-V5 (R960-25) (dilution 1:5,000) from Invitrogen; anti-PARP1 (BML-SA250-0050) from Enzo Life Science; anti-GAPDH (NB300-22) (dilution 1:5,000) from Novus Biologicals; anti-cleaved caspase-8 (9496), anti-CYLD (4495), anti-RIP1 (137451) and anti-p-RIP1(65746) from Cell Signaling Technology; anti-DsRed (632392) (dilution 1:5,000) from Clontech.

Techniques: Immunoprecipitation, shRNA, Transfection

Journal: Investigative Ophthalmology & Visual Science

Article Title: CX3CL1/CX3CR1 Signaling Mediated Neuroglia Activation Is Implicated in the Retinal Degeneration: A Potential Therapeutic Target to Prevent Photoreceptor Death

doi: 10.1167/iovs.65.1.29

Figure Lengend Snippet: CX3CL1-CX3CR1 signaling activation in the retina of RD model. ( A and B ) The number of CX3CL1/CX3CR1 positive cells increased significantly at the onset of RD. ( C and D ) The protein levels of CX3CL1/CX3CR1 increased significantly during the RD process. The expressions of p-NF-κB, a downstream mediator of CX3CL1/CX3CR1 pathway, also increased significantly in the RD model. ( E ) (* P < 0.05, ** P < 0.01, and **** P < 0.0001, for differences between time points, and # P < 0.05, ## P < 0.01, ### P < 0.001, and #### P < 0.0001, for differences between animal groups, n = 6).

Article Snippet: Then, the retinal sections were incubated with primary antibody indulging IBA1 (ab178847, 1:100; Abcam), GFAP (G3893, 1:200; Sigma), Rhodopsin (ab98887, 1:500; Abcam), CX3CL1 (ab25088, 1:500; Abcam), CX3CR1 (2093, 1:500; ProSci), GS (ab176562, 1:250; Abcam), Ly6G (ab25377, 1:100; Abcam,) at 4°C overnight.

Techniques: Activation Assay

Journal: Investigative Ophthalmology & Visual Science

Article Title: CX3CL1/CX3CR1 Signaling Mediated Neuroglia Activation Is Implicated in the Retinal Degeneration: A Potential Therapeutic Target to Prevent Photoreceptor Death

doi: 10.1167/iovs.65.1.29

Figure Lengend Snippet: AZD8797 suppress the CX3CL1/CX3CR1 signaling pathway. ( A and B ) The number of CX3CL1 and CX3CR1 positive cells in the RD + AZD8797 group, reduced significantly compared with the RD group. ( C , D , and E ) Western blot results showed that the protein levels of CX3CL1, CX3CR1, and p-NF-κB in the RD + AZD8797 group reduced significantly compared with the RD group, the expression levels of CX3CL, CX3CR1, and NF-κB protein were analyzed by Western blot assay. NF-κB and statistical line chart of gray value. (Scale = 20 µm; * P < 0.05, ** P < 0.01, and **** P < 0.0001, for differences compared with the normal control, and # P < 0.05, ## P < 0.01, and ### P < 0.001, for differences between animal groups, n = 6).

Article Snippet: Then, the retinal sections were incubated with primary antibody indulging IBA1 (ab178847, 1:100; Abcam), GFAP (G3893, 1:200; Sigma), Rhodopsin (ab98887, 1:500; Abcam), CX3CL1 (ab25088, 1:500; Abcam), CX3CR1 (2093, 1:500; ProSci), GS (ab176562, 1:250; Abcam), Ly6G (ab25377, 1:100; Abcam,) at 4°C overnight.

Techniques: Western Blot, Expressing, Control