Journal: eLife

Article Title: Polymerization of misfolded Z alpha-1 antitrypsin protein lowers CX3CR1 expression in human PBMCs

doi: 10.7554/eLife.64881

Figure Lengend Snippet: Lipid rafts were solubilized from membrane fractions with UltraRIPA kit. ( a ) For analysis of CX3CR1, equal amounts of protein were separated by SDS-PAGE under reducing conditions. One representative blot from n = 3 independent experiments is shown. ( b ) For analysis of lipid raft associated AAT polymers, the same samples were separated under non-reducing conditions. The Western blot was probed with monoclonal antibody (2C1) recognizing polymeric AAT. One representative blot from n = 3 independent experiments is shown.

Article Snippet: Antibody , Anti-AAT polymer (mouse monoclonal) , Hycult Biotech , Clone 2C1; HM2289 , (1:500).

Techniques: SDS Page, Western Blot

Journal: eLife

Article Title: Polymerization of misfolded Z alpha-1 antitrypsin protein lowers CX3CR1 expression in human PBMCs

doi: 10.7554/eLife.64881

Figure Lengend Snippet:

Article Snippet: Antibody , Anti-AAT polymer (mouse monoclonal) , Hycult Biotech , Clone 2C1; HM2289 , (1:500).

Techniques: TaqMan Assay, Purification, Enzyme-linked Immunosorbent Assay, Immunofluorescence, Recombinant, Software

Journal: The Journal of allergy and clinical immunology

Article Title: ORAI1 mutations abolishing store-operated Ca 2+ entry cause anhidrotic ectodermal dysplasia with immunodeficiency (EDA-ID)

doi: 10.1016/j.jaci.2017.10.031

Figure Lengend Snippet: Summary of clinical and laboratory findings in P1–P4.

Article Snippet: For ORAI1 cell surface protein expression, patient and HD control fibroblasts were stained with an Alexa Fluor 647-conjugated anti-human ORAI1 monoclonal antibody (2C1.1) that recognizes an epitope in the second extracellular loop (ECL2) of ORAI1 (kind gift of H. McBride, Amgen).

Techniques: Mutagenesis, Expressing, Infection, Isolation

Journal: The Journal of allergy and clinical immunology

Article Title: ORAI1 mutations abolishing store-operated Ca 2+ entry cause anhidrotic ectodermal dysplasia with immunodeficiency (EDA-ID)

doi: 10.1016/j.jaci.2017.10.031

Figure Lengend Snippet: A–F, Pedigrees (A,C,E) and mRNA/protein sequences of ORAI1 mutations (B,D,F) identified in three unrelated kindreds. A,B, Patient 1 (P1, A-II-1) of kindred A is homozygous for a single nucleotide deletion (c.del541C) in exon 2 of ORAI1 that results in a frameshift and premature stop codon at V181SfsX8 in the third transmembrane domain (TM3) of ORAI1 protein. C,D, P2 (B-II-1) is homozygous for a single nucleotide transition (c.T581C) in exon 2 of ORAI1 that results in a single amino acid substitution in TM3 (p.L194P). E,F, P3 (C-II-2) and his sister P4 (C-II-1) are homozygous for a single nucleotide transversion (c.G292C) in exon 1 of ORAI1 that results in a single amino acid substitution in TM1 (p.G98R). Filled symbols in A, C, E represent patients; dots in empty symbols represent confirmed heterozygous carriers; double lines indicate consanguinity. G, Homology model of the hexameric human ORAI1 protein structure modeled on the D. melanogaster Orai crystal structure 19. Tertiary structure of the ORAI1 hexamer from the side (top) and the extracellular side of the plasma membrane (PM) revealing the channel pore (bottom). The inner (IN) and outer (OUT) leaflets of the PM are indicated; TM domains are color coded (TM1: yellow; TM2 orange; TM3: green; TM4 blue). Amino acid residues mutated in P1–P4 are shown in color (V181: red; L194: magenta; G98: blue). H–I, Structure models of wildtype (top) and mutant (bottom) ORAI1 (H: L194P; I: G98R). The models orient the mutant side chains in positions homologous to the wildtype residues and do not show an experimentally determined structure of mutant proteins.

Article Snippet: For ORAI1 cell surface protein expression, patient and HD control fibroblasts were stained with an Alexa Fluor 647-conjugated anti-human ORAI1 monoclonal antibody (2C1.1) that recognizes an epitope in the second extracellular loop (ECL2) of ORAI1 (kind gift of H. McBride, Amgen).

Techniques: Mutagenesis

Journal: The Journal of allergy and clinical immunology

Article Title: ORAI1 mutations abolishing store-operated Ca 2+ entry cause anhidrotic ectodermal dysplasia with immunodeficiency (EDA-ID)

doi: 10.1016/j.jaci.2017.10.031

Figure Lengend Snippet: A–C, SOCE measurements in fibroblasts of P1, P2, P3 and a HD control (CTRL). Cells were loaded with Fura-2 and stimulated with thapsigargin (TG) in the absence of extracellular Ca2+ followed by readdition of 20 mM Ca2+. Traces show intracellular Ca2+ levels (F340/380) recorded by time-lapse microscopy and represent the average ± SEM of > 64 cells from one representative experiment. D–F, Fibroblasts from P1, P2 and P3 were retrovirally transduced with bicistronic vectors encoding wildtype ORAI1 (IRES-GFP) or STIM1 (IRES-GFP). Intracellular Ca2+ levels in GFP+ fibroblasts were measured as described in A–C. Shown are Ca2+ traces from one representative experiment; > 30 cells were analyzed. G–I, Bar graphs show the mean ± SEM of peak intracellular Ca2+ levels after TG stimulation and readdition of 20 mM Ca2+(left) and the Ca2+ influx rate in the first 20 s after readdition of Ca2+(right). Ca2+ traces in A–F and bar graphs in G–I are representative of two (P2, P3) and three (P1) independent experiments.

Article Snippet: For ORAI1 cell surface protein expression, patient and HD control fibroblasts were stained with an Alexa Fluor 647-conjugated anti-human ORAI1 monoclonal antibody (2C1.1) that recognizes an epitope in the second extracellular loop (ECL2) of ORAI1 (kind gift of H. McBride, Amgen).

Techniques: Time-lapse Microscopy, Transduction

Journal: The Journal of allergy and clinical immunology

Article Title: ORAI1 mutations abolishing store-operated Ca 2+ entry cause anhidrotic ectodermal dysplasia with immunodeficiency (EDA-ID)

doi: 10.1016/j.jaci.2017.10.031

Figure Lengend Snippet: A–C, Bar graphs show the mean ± SEM of ORAI1 mRNA isolated from fibroblasts of P1 (A), P2 (B) and P3 (C) compared to HD control fibroblasts (CTRL) and measured by qPCR. D–F, Flow cytometric analysis of ORAI1 (red) at the surface of fibroblasts from P1 (D), P2 (E), P3 (F) and HD control fibroblasts (CTRL) using an antibody against the second extracellular domain of ORAI1. Unstained fibroblasts were used as control (gray). Bar graphs show the average of ΔMFIs (calculated as MFIORAI1 – MFIunstained control) ± SEM. Data in A–C and D–F represent 2 independent experiments for each patient. Statistical significance was calculated using unpaired Student’s t test, p<0.05*, p<0.01**.

Article Snippet: For ORAI1 cell surface protein expression, patient and HD control fibroblasts were stained with an Alexa Fluor 647-conjugated anti-human ORAI1 monoclonal antibody (2C1.1) that recognizes an epitope in the second extracellular loop (ECL2) of ORAI1 (kind gift of H. McBride, Amgen).

Techniques: Isolation

Journal: The Journal of allergy and clinical immunology

Article Title: ORAI1 mutations abolishing store-operated Ca 2+ entry cause anhidrotic ectodermal dysplasia with immunodeficiency (EDA-ID)

doi: 10.1016/j.jaci.2017.10.031

Figure Lengend Snippet: A, Measurements of SOCE in T cells from P3 and a healthy control (CTRL) by time-lapse microscopy. Cells were stimulated with thapsigargin (TG) followed by readdition of 1.2 mM extracellular Ca2+. Traces show intracellular Ca2+ levels (F340/380) of > 60 cells (average ± SEM). Bar graphs represent the mean ± SEM of the SOCE peak in 1.2 mM Ca2+ (top) and the Ca2+ influx rate in the first 20 s after readdition of Ca2+(bottom). Data are representative for three experiments. B, Cytokine production by PBMC from P6 (ORAI1 p.R91W) 3 isolated at 20 years of age and an adult HD control. PBMC were stimulated with PMA (40 ng/ml) and ionomycin (500 ng/ml) for 4 hours, and analyzed by intracellular cytokine staining and flow cytometry. C–D, Flow cytometry analysis of PBMC from the 20-year-old P6 and a representative HD control for naïve, central memory and effector memory CD4+ and CD8+ T cells (C) and Foxp3+ Treg cells (D). E, Flow cytometry analysis of PBMC from P3 (ORAI1 p.G98R) and a HD control for Foxp3+ Treg cells. Contour plots are gated on CD4+ cells. Numbers in red in panels B–E indicate marked differences in T cell subsets in P6 compared to HD controls.

Article Snippet: For ORAI1 cell surface protein expression, patient and HD control fibroblasts were stained with an Alexa Fluor 647-conjugated anti-human ORAI1 monoclonal antibody (2C1.1) that recognizes an epitope in the second extracellular loop (ECL2) of ORAI1 (kind gift of H. McBride, Amgen).

Techniques: Time-lapse Microscopy, Isolation, Staining, Flow Cytometry