Journal: Stem cells (Dayton, Ohio)

Article Title: Effect of inflammatory cytokines on major histocompatibility complex expression and differentiation of human neural stem/progenitor cells.

doi: 10.1634/stemcells.2008-0116

Figure Lengend Snippet: Figure 5. Expression of cytokine receptors in human neural stem/progenitor cell (hNSPC) cultures. Cultures of hNSPCs were immunohistochemically labeled for cytokine receptors (A–E), and the expression levels on the extracellular membrane were deter- mined by luminosity measurements (F, G) and expressed as luminosity units (LU)/cell. (F): Undifferentiated hippocampal line HPC0A07 (HPC) cells expressed higher lev- els of the two interferon--R (IFN--R) sub- units and tumor necrosis factor--R1 (TNF- -R1) compared with striatal cell line STROC05 (STR) cells. (G): Except for a slightly higher expression of interleukin-6-R (IL-6-R) on HPC cells, there was no differ- ence in receptor expression level after dif- ferentiation. All photomicrographs were captured at 40. (A–E): Scale bar 25 m. (A–D): Error bars: mean SEM. , p .05; , p .01; , p .005.

Article Snippet: Furthermore, to determine whether the cells expressed receptors for the different cytokines, cultures were single-labeled for IFN -R (rabbit anti-IFN -R , 1:200), IFN -R (rabbit anti-IFN -R , 1:200), TNF -R1 (mouse anti-TNF -R1, 1:200), TNF -R2 (goat antiTNF -R2, 1:200), or IL-6-R (rabbit anti-IL6-R, 1:500), all from Santa Cruz Biotechnology Inc., Santa Cruz, CA, http://www.scbt. com.

Techniques: Expressing, Labeling, Membrane

Journal: Journal of Biological Chemistry

Article Title: A Death Receptor-associated Anti-apoptotic Protein, BRE, Inhibits Mitochondrial Apoptotic Pathway

doi: 10.1074/jbc.m408678200

Figure Lengend Snippet: FIG. 4. BRE associates with TNF-R1 and Fas. A, co-immunoprecipitation of TNF-R1 with GS-BRE and V5-BRE using anti-V5 monoclonal antibody from HeLa transiently transfected with the respec- tive expression constructs. MAB2, a third party mouse monoclonal antibody against phosphorylcholine, is the negative control. Immunoblotting was performed using a rabbit anti-TNF-R1 polyclonal antibody (Calbiochem). B, co-immunoprecipitation of GS-BRE (52 kDa) with TNF-R1 and Fas using mouse anti-TNF-R1 monoclonal antibody (clone 16803, R & D Systems) and rabbit anti-Fas polyclonal antibodies (C20, Santa Cruz Biotechnology), respectively. MAB2, rat anti-TGF monoclonal antibody (clone A75-2.1, Pharmingen), and normal rabbit serum are negative controls. GS- BRE was immunoblotted by HRP-conju- gated anti-V5 monoclonal antibody. C, co- immunoprecipitation of Fas with GS-BRE detected by rabbit anti-Fas polyclonal an- tibody (C20). Two monoclonal anti-Fas antibodies (clone DX2, Pharmingen, and clone 13, Transduction Laboratories) are positive controls for Fas immunoprecipi- tation. D, co-precipitation of GS-BRE with Fas immunoprecipitated by 2 anti- Fas antibodies, DX2 and C20. GS-BRE was immunoblotted by HRP-conjugated anti-V5 monoclonal antibody. IP, immu- noprecipitation; WB, Western blotting analysis.

Article Snippet: Reagents—Antibodies (clone or code number) and reagents were purchased from the following sources: rabbit polyclonal anti-TNF-R1 and anti-ubiquitin antibodies (Calbiochem); rabbit polyclonal anti-Fas (C-20), anti-TRADD (H-278), and mouse monoclonal anti-SUMO-1 (D11) antibodies (Santa Cruz Biotechnology, Santa Cruz, CA); mouse monoclonal anti-GAPDH (9.B.88) and anti- -actin (2A2.1) antibodies (U. S. Biological, Swampscott, MA); mouse monoclonal anti-Fas (13), anti-G28, and anti-BiP antibodies (Transduction Laboratories); mouse monoclonal anti-Fas (DX2), anti-cytochrome c (7H8.2C12), rat monoclonal anti-TGF (A75-2.1), rabbit polyclonal anti-BID, and anticaspase-8 (poly-1326) antibodies (Pharmingen); rabbit polyclonal anticleaved caspase-3, anti-cleaved caspase-9, anti-PARP, and mouse monoclonal anti-caspase-8 (1C12) antibodies (Cell Signaling Technology, Beverly, MA); mouse monoclonal anti-Fas IgM antibody (CH11), anti-Smac/DIABLO (78-1-118), and anti-histone H1 (AE-4) antibodies (Upstate Biotechnology, Inc.); rabbit polyclonal anti-pan-phosphoprotein and mouse monoclonal anti- -tubulin (2-28-33) antibodies (Zymed Laboratories Inc.); rabbit polyclonal anti-catalase antibody (Abcam Ltd., Cambridge, UK); mouse monoclonal anti-prohibitin (Ab-1) antibody (Lab Vision Corp., Fremont, CA); mouse monoclonal anti-TNF-R1 antibodies (16803) with and without FITC conjugation, and goat antiTNF-R1 affinity-purified polyclonal antibody (R&D Systems, Minneapolis, MN); mouse monoclonal anti-V5 antibody and the horseradish peroxidase (HRP) conjugate (Invitrogen); HRP-conjugated anti-rabbit Ig secondary antibody (Promega, Madison, WI); FITC-conjugated antirabbit, anti-mouse, HRP-conjugated anti-mouse Ig secondary antibodies, and protease inhibitors mixture (Sigma); human recombinant TNF- (Invitrogen); -protein phosphatase (New England Biolabs, Beverly, MA); GS-BRE (ResGen GeneStorm® Clone for accession number L38616), GS alone expression vector, G418, Zeocin, and Lipofectamine 2000 (Invitrogen).

Techniques: Immunoprecipitation, Transfection, Expressing, Construct, Negative Control, Western Blot, Transduction

Journal: Journal of Biological Chemistry

Article Title: A Death Receptor-associated Anti-apoptotic Protein, BRE, Inhibits Mitochondrial Apoptotic Pathway

doi: 10.1074/jbc.m408678200

Figure Lengend Snippet: FIG. 6. BRE dissociates from TNF-R1, but not from Fas, upon receptor ligation. NHGS2 was treated with 100 ng/ml TNF- (A) or 200 ng/ml CH11 (B). At the indicated time points, cell lysates of A were subjected to immunoprecipitation (IP) by mouse monoclonal anti- TNF-R1 antibody (clone 16803, R&D Systems), and the precipitates were immunoblotted with anti-V5-HRP, rabbit anti-TRADD, and TNF-R1 polyclonal antibodies. The same result was obtained by using a goat anti-TNF-R1 affinity-purified polyclonal antibody (R&D Sys- tems) for immunoprecipitation. Cell lysates of B were subjected to immunoprecipitation by monoclonal anti-Fas (DX2) antibody, and the precipitates were immunoblotted with anti-V5-HRP, anti-caspase-8, and Fas (C20) antibodies. Immunoprecipitation by anti-GAPDH anti- body is the negative control. All the cell lysates were also immuno- blotted directly with anti-V5-HRP for GS-BRE to ensure similar amount of starting proteins for immunoprecipitation and to show no degradation of GS-BRE during the time course study.

Article Snippet: Reagents—Antibodies (clone or code number) and reagents were purchased from the following sources: rabbit polyclonal anti-TNF-R1 and anti-ubiquitin antibodies (Calbiochem); rabbit polyclonal anti-Fas (C-20), anti-TRADD (H-278), and mouse monoclonal anti-SUMO-1 (D11) antibodies (Santa Cruz Biotechnology, Santa Cruz, CA); mouse monoclonal anti-GAPDH (9.B.88) and anti- -actin (2A2.1) antibodies (U. S. Biological, Swampscott, MA); mouse monoclonal anti-Fas (13), anti-G28, and anti-BiP antibodies (Transduction Laboratories); mouse monoclonal anti-Fas (DX2), anti-cytochrome c (7H8.2C12), rat monoclonal anti-TGF (A75-2.1), rabbit polyclonal anti-BID, and anticaspase-8 (poly-1326) antibodies (Pharmingen); rabbit polyclonal anticleaved caspase-3, anti-cleaved caspase-9, anti-PARP, and mouse monoclonal anti-caspase-8 (1C12) antibodies (Cell Signaling Technology, Beverly, MA); mouse monoclonal anti-Fas IgM antibody (CH11), anti-Smac/DIABLO (78-1-118), and anti-histone H1 (AE-4) antibodies (Upstate Biotechnology, Inc.); rabbit polyclonal anti-pan-phosphoprotein and mouse monoclonal anti- -tubulin (2-28-33) antibodies (Zymed Laboratories Inc.); rabbit polyclonal anti-catalase antibody (Abcam Ltd., Cambridge, UK); mouse monoclonal anti-prohibitin (Ab-1) antibody (Lab Vision Corp., Fremont, CA); mouse monoclonal anti-TNF-R1 antibodies (16803) with and without FITC conjugation, and goat antiTNF-R1 affinity-purified polyclonal antibody (R&D Systems, Minneapolis, MN); mouse monoclonal anti-V5 antibody and the horseradish peroxidase (HRP) conjugate (Invitrogen); HRP-conjugated anti-rabbit Ig secondary antibody (Promega, Madison, WI); FITC-conjugated antirabbit, anti-mouse, HRP-conjugated anti-mouse Ig secondary antibodies, and protease inhibitors mixture (Sigma); human recombinant TNF- (Invitrogen); -protein phosphatase (New England Biolabs, Beverly, MA); GS-BRE (ResGen GeneStorm® Clone for accession number L38616), GS alone expression vector, G418, Zeocin, and Lipofectamine 2000 (Invitrogen).

Techniques: Ligation, Immunoprecipitation, Affinity Purification, Negative Control