Journal: European journal of immunology

Article Title: Comparison of stable human Treg and Th clones by transcriptional profiling.

doi: 10.1002/eji.200838807

Figure Lengend Snippet: Figure 4. Human Treg clones, but not Th clones, produce the mature and active form of TGF-b. (A) Schematic representation of TGF-b processing. Double lines represent cell membrane. Sites of proteolytic cleavages are indicated by arrow heads. Small bars indicate disulfide bonds. LAP: latency associated peptide. LTBP: latent TGF-b binding protein; TGFBR: TGF-b receptors. (B) Three Treg clones (Treg D1, Treg B2, and Treg B3) and four Th clones (Th C2, Th D1, Th B1, and Th A2) were activated in X-VIVO-10 serum-free medium for 24 h with anti-CD3 and anti-CD28 Ab. Active and total TGF-b concentrations in the supernatants were measured by ELISA, before or after treatment with acid, respectively. Values represent means of duplicate1SD. (C) Four Treg clones (Treg C2, Treg D1, Treg B2, and Treg B3) and four Th clones (Th C2, Th D1, Th B1, and Th A2) were activated for 24 h with anti-CD3 and anti-CD28 Ab in the presence of IL-2, prior to staining with biotinylated polyclonal anti-LAP antibody (filled gray histograms) or isotype control (empty histograms) and Streptavidin-PE. (D) Western blot analysis of cell lysates from Treg and Th clones collected at rest (T 5 0), or 6 or 24 h after activation with anti-CD3 and anti-CD28 Ab. Samples in lanes 4 and 8 were treated with 5 ng/mL of recombinant human TGF-b1 (rhTGF-b1) during the last 15 min of the activation period and serve as positive controls. Samples in lanes 17 and 18 were prepared as detailed in Fig. 3D. Briefly, suppressed (‘‘S’’) or control (‘‘Ctrl’’) clone Th A2 was sorted by FACS out of a co-culture with clone Treg A1 or clone Th A2, respectively. Blots were hybridized with an anti-phosphorylated SMAD2 (P-SMAD2) or anti-b-actin antibody.

Article Snippet: For surface LAP expression, cells activated for 24 h with anti-CD3 and anti-CD28 antibodies in the presence of IL-2 (160 IU/mL) were labeled with a biotinylated polyclonal anti-LAP antibody or an isotype control (R&D Systems, BAF246 and BAF108, respectively), followed by incubation with Streptavidin coupled to PE (BD Pharmingen).

Techniques: Clone Assay, Membrane, Binding Assay, Enzyme-linked Immunosorbent Assay, Staining, Control, Western Blot, Activation Assay, Recombinant, Co-Culture Assay

Journal: Brain, behavior, and immunity

Article Title: Reducing age-dependent monocyte-derived macrophage activation contributes to the therapeutic efficacy of NADPH oxidase inhibition in spinal cord injury

doi: 10.1016/j.bbi.2018.11.013

Figure Lengend Snippet: Antibodies Used

Article Snippet: Biotinylated goat anti-chicken (IgY) Goat 1:1000 Aves Lab B-1005 8.

Techniques: Immunohistochemistry

Journal: Journal of neurochemistry

Article Title: Developmental alteration of nerve injury induced glial cell line-derived neurotrophic factor (GDNF) receptor expression is crucial for the determination of injured motoneuron fate.

doi: 10.1046/j.1471-4159.2002.01043.x

Figure Lengend Snippet: Fig. 2 Expression of GDNF receptors mRNAs in injured motoneurons during the course of postnatal development. (a) Film autoradiography demonstrated that altera- tion of GFRa1 and c-Ret mRNA expression in the injured hypoglossal nucleus. 4P3, 7P3, 8P3, 11P3, 12P3 and 35P3 indicate that rats were axotomized on 4, 7, 8, 11, 12 and 35 days after birth, respectively, and then killed on postoperative day 3. The arrows indicate the axotomized side of nuclei. Marked conversion from down- to up-regulation of GFRa1 mRNA expression in injured side hypoglossal nucleus was observed along with the development, whereas c-Ret mRNA expression in injured motoneurons is up-regulated in both neo- nates and adults. Scale bar ¼ 500 lm. (b) Immunohistochemical demonstrations using anti-GFRa1 (left column) and anti-c- Ret antibody (right column) in injured motoneurons during the course of devel- opment. 3P3, 7P3, 14P3, 42P3 indicate that rats were axotomized on 3, 7, 14 and 42 days after birth, respectively, and killed 3 days after the axotomy. Scale bar ¼ 250 lm. (c) Semi-quantification of GFRa1 and c-Ret mRNA expression in injured motoneurons. Vertical axis indicates the optical intensity ratio (O/C, see Materials and methods) and the horizontal axis indi- cates the axotomized age. Each point shows the average of the intensity of the positive signal ratios and error bars indicate SE. The dots in the shaded area of the graph mean its expression in injured moto- neuron was down-regulated (O/C < 1).

Article Snippet: Goat anti-GDNF polyclonal antibody (1 : 50; R & D Systems, Minneapolis, MN, USA), biotinylated goat anti-GFRa1 polyclonal antibody (1 : 50, R & D) and rabbit anti-c-Ret polyclonal antibody, R787 (1 : 50, IBL, Fujioka, Japan) were used for detection of GDNF, GFRa1 and c-Ret, respectively.

Techniques: Expressing, Autoradiography, Immunohistochemical staining