Journal: Nature medicine

Article Title: An AKT3-FOXG1-Reelin Network Underlies Defective Migration in Human Focal Malformations of Cortical Development

doi: 10.1038/nm.3982

Figure Lengend Snippet: ( a ) Quantitative real-time PCR was performed to measure RELN expression in hNPCs expressing AKT3 E17K and compared to vector-expressing hNPCs. Three independent experiments were quantified in triplicate. ( b ) Mouse embryos at E14.5 were electroporated with indicated DNA constructs. Brains isolated at E18.5 used for reelin expression. Arrowheads: reelin immunostaining in pericellular area of ectopic neurons (intensity-per-pixel: 622.55 ± 45.80 compared to 4092.77 ± 9.46, marginal zone). ( c ) E14.5 embryos were co-electroporated with indicated constructs or RNAs. ( d ) Localization of GFP + neurons at E18.5 was quantified in each cortical region ( n = 3 for each). ( e, f ) Developing cortices were in utero electroporated (IUE) at E14.5 with RFP vector. After 15 min, embryos were sequentially electroporated with either GFP vector ( n = 3) or GFP also expressing AKT3 E17K (A3) with or without Reln siRNA ( n = 6 and 4, respectively). At E18.5, brains were sectioned for imaging. Localization of RFP + GFP − cells was quantified in each cortical region. Values: mean ± s.d. n.s., not significant; **, P < 0.01; ***, P < 0.001, Student’s t -test ( a ); G-test of goodness-of-fit ( d, f ). Scale bars: 25 μm ( b ); 100 μm ( c, e ). ( g ) Enrichment of FOX-binding site. See Online Methods for details. ( h ) Model of effect of AKT3 mutation on surrounding cells. Mutant cell, pink; surrounding cells, gray. AKT3* activating somatic mutation leads to mTOR activation and phosphorylation and cytoplasmic sequestration of FOXG1, relieving repression of RELN , which is then secreted by mutant cells to act in an autocrine (cell autonomous) and paracrine (non-cell autonomous) fashion.

Article Snippet: Paraffin embedded tissues were sectioned, rehydrated and retrieved for antigen (citrated buffer, pH 6.0) before immunostaining for GFP (1:200; Rockland; cat. no. 600-106-215) followed by detection by DAB (Vector Labs).

Techniques: Real-time Polymerase Chain Reaction, Expressing, Plasmid Preparation, Construct, Isolation, Immunostaining, In Utero, Imaging, Binding Assay, Mutagenesis, Activation Assay, Phospho-proteomics

Journal: The Journal of investigative dermatology

Article Title: α(1,3) Fucosyltransferases IV and VII are essential for the initial recruitment of basophils in chronic allergic inflammation.

doi: 10.1038/jid.2013.160

Figure Lengend Snippet: Figure 4. Selectin-dependent cooperative recruitment of basophils and effector cells. (a) Irradiated a(1, 3) fucosyltransferase-IV (FT-IV)( / )/FT-VII( / ) mice received wild-type (WT) basophils in combination with CD49b( ) bone marrow cells (effector cells) from either WT or FT-IV( / )/FT-VII( / ) mice. They were then immunized with trinitrophenyl-IgE (TNP-IgE) and challenged with TNP-OVA. The untreated group comprised FT-IV( / )/FT-VII( / ) mice without cell transfer. (b) Cell populations in inflammatory skin. *Po0.05 compared with WT effector cells. BM, basement membrane.

Article Snippet: In vitro selectin binding assay of basophils CD49b (þ ) basophil–enriched bone marrow cells were suspended in phosphate-buffered saline containing 5% fetal calf serum, 0.1% NaN3, 1 mmol l 1 Ca2þ , and 1 mmol l 1 Mg2þ , followed by incubation with 10mg ml 1 of murine P-, E-, or L-selectin-human IgG Fc chimera or control human IgG1 Fc (R&D Systems) for 40 minutes at 4 1C.

Techniques: Irradiation, Membrane

Journal: The Journal of investigative dermatology

Article Title: α(1,3) Fucosyltransferases IV and VII are essential for the initial recruitment of basophils in chronic allergic inflammation.

doi: 10.1038/jid.2013.160

Figure Lengend Snippet: Figure 5. Expression of a(1, 3) fucosyltransferase (FT) messenger RNA (mRNA) and FT-dependent selectin binding in basophils. (a) Primary basophils were subjected to further purification by positive selection with CD123 (purity 499%). Transcripts for FT-IV and FT-VII mRNA were quantified by real-time PCR. (b) Binding of soluble P- and E-selectins to primary basophils assessed by flow cytometry. BMBa, bone marrow–derived basophil; BMMCs, bone marrow–derived mast cells.

Article Snippet: In vitro selectin binding assay of basophils CD49b (þ ) basophil–enriched bone marrow cells were suspended in phosphate-buffered saline containing 5% fetal calf serum, 0.1% NaN3, 1 mmol l 1 Ca2þ , and 1 mmol l 1 Mg2þ , followed by incubation with 10mg ml 1 of murine P-, E-, or L-selectin-human IgG Fc chimera or control human IgG1 Fc (R&D Systems) for 40 minutes at 4 1C.

Techniques: Expressing, Binding Assay, Selection, Real-time Polymerase Chain Reaction, Cytometry, Derivative Assay

Journal: The Journal of investigative dermatology

Article Title: α(1,3) Fucosyltransferases IV and VII are essential for the initial recruitment of basophils in chronic allergic inflammation.

doi: 10.1038/jid.2013.160

Figure Lengend Snippet: Figure 6. Basophil P-selectin glycoprotein-1 (PSGL-1)–L-selectin interaction involvement in IgE-mediated chronic allergic inflammation (IgE-CAI). (a) PSGL-1 and L-selectin expressions on basophils. (b) PSGL-1 Ab effect on selectin binding to primary basophils. (c) L-selectin binding to basophils from wild-type (WT) and a(1, 3) fucosyltransferase-IV (FT-IV)( / )/FT-VII( / ) mice. (d) Effects of L-selectin and/or PSGL-1 Abs on IgE-CAI in WT mice. *Po0.05 compared with control IgG. **Po0.05 compared with L-selectin or PSGL-1 Ab alone.

Article Snippet: In vitro selectin binding assay of basophils CD49b (þ ) basophil–enriched bone marrow cells were suspended in phosphate-buffered saline containing 5% fetal calf serum, 0.1% NaN3, 1 mmol l 1 Ca2þ , and 1 mmol l 1 Mg2þ , followed by incubation with 10mg ml 1 of murine P-, E-, or L-selectin-human IgG Fc chimera or control human IgG1 Fc (R&D Systems) for 40 minutes at 4 1C.

Techniques: Binding Assay, Control