Journal: Differentiation; research in biological diversity

Article Title: Transformation of jaw muscle satellite cells to cardiomyocytes

doi: 10.1016/j.diff.2016.11.003

Figure Lengend Snippet: A. Scheme of the growth and differentiation protocol. B. Percentage of cTnT-positive cells generated by the end of the differentiation period, with and without serum. Error bars indicate ± standard error of the mean. C. qRT-PCR of expression of myogenic and cardiogenic transcription factors during the first 7 days. Error bars indicate ± standard error of the mean. D. NKX2.5 (red) is found only in those cells expressing PAX7 (green) at day 3 cultures. Scale bar 25μm. E. At 7 days of culture, myogenin (green) is not co-expressed with GATA4 (red). Scale bar 25μm.

Article Snippet: Briefly, slides were washed in 1xPBSA, permeabalized in PBSA containing 0.1% Triton, blocked in 5% normal goat serum (NGS) for 2 hours at RT and incubated overnight with primary antibodies to either PAX7 (1/1000; Developmental Studies Hybridoma Bank, DSHB), MHC (MF20; 1/500; DSHB), NKX2.5 (1/200; Santa Cruz), GATA4 (1/500; ABCAM), Myogenin (F5D, 1:100; DSHB), cTnT (CT-3; 1/500; DSHB) in 5% NGS at 4°C.

Techniques: Generated, Quantitative RT-PCR, Expressing

Journal: Differentiation; research in biological diversity

Article Title: Transformation of jaw muscle satellite cells to cardiomyocytes

doi: 10.1016/j.diff.2016.11.003

Figure Lengend Snippet: A. Evidence that induced cardiomyocytes are derived from satellite cells. Pax7 expressing cells were labeled by treating the Pax7-CreER;mT/mG mice with tamoxifen. The green label indicates that cTnT-positive cardiomyocytes formerly expressed Pax7. Scale bar 25μm. B. Evidence that induced cardiomyocytes are derived from the secondary heart field. Satellite cells were prepared from Isl1-Cre;mTmG mice and then sorted for GFP expression, and subjected to the cardiomyocyte differentiation procedure. The NKX2.5 and cTnT-positive cells must be derived from cells that had expressed Isl1 in vivo. Scale bar: 25μm.

Article Snippet: Briefly, slides were washed in 1xPBSA, permeabalized in PBSA containing 0.1% Triton, blocked in 5% normal goat serum (NGS) for 2 hours at RT and incubated overnight with primary antibodies to either PAX7 (1/1000; Developmental Studies Hybridoma Bank, DSHB), MHC (MF20; 1/500; DSHB), NKX2.5 (1/200; Santa Cruz), GATA4 (1/500; ABCAM), Myogenin (F5D, 1:100; DSHB), cTnT (CT-3; 1/500; DSHB) in 5% NGS at 4°C.

Techniques: Derivative Assay, Expressing, Labeling, In Vivo

Journal: The Journal of Biological Chemistry

Article Title: Single-cell RNA-Seq analysis identifies a noncoding interleukin 4 ( IL-4 ) RNA that post-transcriptionally up-regulates IL-4 production in T helper cells

doi: 10.1074/jbc.RA118.004111

Figure Lengend Snippet: Single-cell RNA-Seq reveals transcriptome heterogeneity of in vitro differentiated Th2 cells. Naïve CD4+ T cells (CD4+CD62LhiCD44lowCD25−) isolated from C57BL/6 mice were stimulated with anti-CD3 and anti-CD28 in the presence of IL-4 and anti-IFN-γ. Five days after stimulation, the cells were restimulated with plate-bound anti-CD3 and anti-CD28 for 6 h, and single cells were captured with a Fluidigm C1 system. A total of 56 cells were collected and sequenced with an Illumina Hiseq system, and 10,000 pooled cells were sequenced using a standard protocol (Pooled cells). A, correlations of gene expression between the pooled cells and average of the 56 single cells (left panel), between two randomly selected single cells (center panel), and between the pooled cells and a single cell (right panel). 10,565 genes were detected. Gene expression was determined by TPM, and Pearson correlation coefficient (r) is depicted. B, heatmap depicting Pearson correlation coefficients between global gene expression profiles of each pair of the 56 individual cells. C, cytokine genes were abundantly expressed and variable among Th2 single cells. Average expression (μ, x axis) and standard deviation (σ, y axis) of each gene were calculated and plotted. The blue dashed lines represent the threshold of CV (highly variable, >0.28; less variable, <0.2). The black dashed line represents the threshold of abundantly expressed genes (μ > 8). Red represents cytokine genes, green denotes housekeeping genes, and gray represents other genes. D, gene ontology (GO) analysis of the most variably expressed genes (μ > 8 and CV > 0.28, left panel) and most consistently expressed genes (μ > 8 and CV < 0.2, right panel). The top five most significantly enriched GO terms (biological processes and molecular functions) are shown. The p values were Bonferroni-corrected.

Article Snippet: Five days after stimulation, the cells were restimulated with plate-bound anti-CD3 and anti-CD28 for 6 h, and single cells were captured with a Fluidigm C1 system.

Techniques: RNA Sequencing Assay, In Vitro, Isolation, Expressing, Standard Deviation

Journal: The Journal of Biological Chemistry

Article Title: Single-cell RNA-Seq analysis identifies a noncoding interleukin 4 ( IL-4 ) RNA that post-transcriptionally up-regulates IL-4 production in T helper cells

doi: 10.1074/jbc.RA118.004111

Figure Lengend Snippet: IL4nc is constitutively expressed in Th2 cells. A, IL4nc was specifically expressed in Th2 and Th0 cells. Naïve CD4+ T cells were activated with anti-CD3 and anti-CD28 and differentiated into various lineages under the respective conditions. Five days after differentiation, expression of IL4nc was analyzed by real-time PCR either under steady state or after being activated by anti-CD3 and anti-CD28 or PAM and ionomycin for 5 h. B, IL4nc is primarily located in the cytoplasm. D10.G4.1 cells were fractionated into the nuclear (N) fraction and cytoplasmic (C) fraction, and the presence of the IL4nc isoform was determined by real-time PCR (left panel). The protein lysates from both fractions were further analyzed by immunoblotting for the cytoplasmic marker (B-actin) and nucleus marker (histone H3) (right panel). C, expression of either IL4nc or IL4fl in resting in vitro differentiated Th2 cells was determined by real-time PCR. D, induction of IL4nc RNA precedes IL4fl following TCR stimulation. Th2 cells were differentiated as described and restimulated with anti-CD3/anti-CD28 for the indicated time. Expression of either IL4nc or IL4fl was determined by real-time PCR. Data are representative (B) or the sum (A–D) of at least three independent experiments. Error bars stand for the standard deviation of the mean. **, p < 0.01 in paired Student's t test. ns, not significantly changed.

Article Snippet: Five days after stimulation, the cells were restimulated with plate-bound anti-CD3 and anti-CD28 for 6 h, and single cells were captured with a Fluidigm C1 system.

Techniques: Expressing, Real-time Polymerase Chain Reaction, Western Blot, Marker, In Vitro, Standard Deviation

Journal: The Journal of Biological Chemistry

Article Title: Single-cell RNA-Seq analysis identifies a noncoding interleukin 4 ( IL-4 ) RNA that post-transcriptionally up-regulates IL-4 production in T helper cells

doi: 10.1074/jbc.RA118.004111

Figure Lengend Snippet: IL4nc RNA promotes IL-4 production in Th2 cells. A, a diagram showing the gene structure of IL4fl (top panel) or IL4nc RNAs (bottom panel). The gray boxes depict noncoding region of transcripts, and the black boxes depict coding regions. B and C, overexpression of IL4nc promotes IL-4 production in Th2 cells. Th2 cells were differentiated in vitro as described in Fig. 1 and transduced with a retrovirus containing either IL4nc RNA or empty vector (Mock). 5 days after stimulation, the cells were restimulated with PMA (50 ng/ml) and ionomycin (500 ng/ml) for 5 h. Expression of IL-4 was determined by intracellular staining. Data shown are representative (B) or summary (C) of IL-4+% or IL-13+% cells from three independent experiments. D, Th2 cells were differentiated and stimulated as in B, and secreted IL-4 in the supernatant was determined by ELISA. Data are summary of three independent experiments. E–G, exon 3 of IL4nc is required for promoting IL-4 production post-transcriptionally. As above, full-length IL4nc (FL) or its mutants depleted the indicated exons (Δexon1, Δexon2, and Δexon3, respectively) and were retrovirally expressed in Th2 cells. Production of IL-4 was analyzed by intracellular staining (E and F) or ELISA (G). H, as in E. Expression of IL-4 and GATA3 mRNAs 2 h after restimulation was analyzed by real-time quantitative PCR. I, as in B. Th2 cells were transduced with a retrovirus overexpressing IL4nc RNA, and the cells were selected with puromycin for 3 days (3 μg/ml). 5 days after differentiation, expression of IL4fl was analyzed by real-time PCR after activation by anti-CD3 (5 μg/ml) and anti-CD28 (1 μg/ml) (left panel) or PMA (50 ng/ml) and ionomycin (500 ng/ml) (right panel) for the indicated time. Data are representative (B and E) or the sum (C, D, and F–I) of at least three independent experiments. Error bars stand for the standard deviation of the mean. *, p < 0.05; **, p < 0.01; paired Student's t test. ns, not significantly changed.

Article Snippet: Five days after stimulation, the cells were restimulated with plate-bound anti-CD3 and anti-CD28 for 6 h, and single cells were captured with a Fluidigm C1 system.

Techniques: Over Expression, In Vitro, Transduction, Plasmid Preparation, Expressing, Staining, Enzyme-linked Immunosorbent Assay, Real-time Polymerase Chain Reaction, Activation Assay, Standard Deviation

Journal: The Journal of Biological Chemistry

Article Title: Single-cell RNA-Seq analysis identifies a noncoding interleukin 4 ( IL-4 ) RNA that post-transcriptionally up-regulates IL-4 production in T helper cells

doi: 10.1074/jbc.RA118.004111

Figure Lengend Snippet: Knockdown of IL4nc RNA represses IL-4 production. Th2 cells were differentiated in vitro as described in Fig. 1 and transduced with a retrovirus containing a scramble sequence (sh-Ctl) or two independent shRNAs against IL4nc RNA. Five days after differentiation, the cells were restimulated with PMA (50 ng/ml) and ionomycin (500 ng/ml) for 5 h. A–C, production of IL-4 was determined by intracellular staining (A and B) and ELISA (C). D, as in A. The cells were restimulated with anti-CD3 and anti-CD28, and expression of IL4fl (left panel) and IL4nc (right panel) RNA was analyzed by real-time PCR at the indicated time after restimulation. E, as in A. 5 days after differentiation, the cells were restimulated with PMA (50 ng/ml) and ionomycin (500 ng/ml) for 3.5 h. The cells were then treated with cycloheximide (100 μg/ml), lysed, and fractionated on a 10–50% sucrose gradient. Distribution of IL4 mRNA at each fraction was analyzed by real-time PCR. Distribution of ribosomal RNA is shown in Fig. S6. F, as in E. In vitro differentiated Th2 cells were transduced with a retrovirus overexpressing IL4nc (IL4nc) or an empty vector (Mock). Distribution of IL4 mRNA at each fraction after sucrose density gradient centrifugation was determined by real-time PCR. Data are representative (A) or the sum (B–F) of at least three independent experiments. Error bars stand for the standard deviation of the mean. *, p < 0.05; **, p < 0.01; paired Student's t test.

Article Snippet: Five days after stimulation, the cells were restimulated with plate-bound anti-CD3 and anti-CD28 for 6 h, and single cells were captured with a Fluidigm C1 system.

Techniques: In Vitro, Transduction, Sequencing, Staining, Enzyme-linked Immunosorbent Assay, Expressing, Real-time Polymerase Chain Reaction, Plasmid Preparation, Gradient Centrifugation, Standard Deviation

Journal: Nano-micro letters

Article Title: Dual-Wavelength Photosensitive Nano-in-Micro Scaffold Regulates Innate and Adaptive Immune Responses for Osteogenesis.

doi: 10.1007/s40820-020-00540-z

Figure Lengend Snippet: Fig. 5 a Schematic diagram of BCP-GNCs-mediated macrophage polarization and DC maturation by releasing IL-4 and DXMS. b IF staining of Arg1 (red) and iNOS (green) of macrophage, CD83 (red) and CD40 (green) of DC and nuclei (blue) after macrophages or DCs seeded on the BCP with 690 nm far-red or 808 nm NIR stimulating the release of PBS (BCP-PBS), 690 nm far-red stimulating the release of IL-4 (BCP-IL4) or 808 nm NIR stimulating the release of DXMS (BCP-DXMS) for 24 h. Scale bar = 5 μm. c, d Semiquantification of positively stained cells in (b). n = 5, **P < 0.01, ***P < 0.001. e Flowchart of time course for irradiating BCP-GNC in vivo. f–h IHC staining of M1, M2 macrophages (iNOS and Arg1), mature DCs (CD40), T cells (CD3), MSCs and osteoblasts (CD146, Runx2) under the BCP-GNCs implant in vivo. Scale bar = 100 μm. Red arrow, positive cells. M, material. i–k Semiquantification of positively stained cells in (E, F, G). n = 5, *P < 0.05, **P < 0.01, ****P < 0.0001. l H&E and Masson staining of implant area of BCP-Con and BCP-GNCs in vivo after 4 weeks (4 W) (the red dash line shows the new bone formation area; NB, new bone; M, material). m Semiquantification of new bone area in (k). n = 5, ****P < 0.0001

Article Snippet: The primary antibodies were listed as: CD11c (1:200 for IF and 1:350 for IHC, Cat: 97,585, CST, USA), F4/80 (1:250, Cat: 70,076, CST, USA), Col1a1(1:100, Cat: SA2005, Boster, China), Runx2 (1:200, Cat: ab76956, Abcam, USA), CD146 (1:100, Cat: A13927, ABclonal, USA), CD83 (1:100, Cat: A2040, ABclonal, USA), CD40 (1:100, Cat: A0218, ABclonal, USA), iNOS (1:100, Sant Cruz, USA.), Arg1 (1:150, Cat: https://doi.org/10.1007/s40820-020-00540-z© The authors A11925, ABclonal, USA), CD3 (1:100, Cat: PB0112, Boster, China).

Techniques: Staining, In Vivo, Immunohistochemistry

Journal: Nano-micro letters

Article Title: Dual-Wavelength Photosensitive Nano-in-Micro Scaffold Regulates Innate and Adaptive Immune Responses for Osteogenesis.

doi: 10.1007/s40820-020-00540-z

Figure Lengend Snippet: Fig. 6 a Implementation strategy of clodronate on macrophages depletion. b H&E and Masson staining of BCP implant area treating with PBS or clodronate in vivo after implant 4 weeks (the red dash line shows the new bone formation area; NB, new bone; M, material). Scale bar = 100 μm. c IHC staining of M2 macrophages (Arg1) and osteoblasts (Runx2, Col1a1) under the BCP implant treating with PBS or clo- dronate in vivo. Scale bar = 100 μm. Red arrow, positive cells. M, material. d–g Semiquantification of new bone area and positively stained cells in (b, c). n = 5, ***P < 0.001, ****P < 0.0001. h Implementation strategy of diphtheria toxin (DT) on DCs depletion. i H&E and Masson stain- ing of BCP implant area treating with PBS or DT in vivo after implant 4 weeks (the red dash line shows the new bone formation area; NB, new bone; M, material). Scale bar = 100 μm. j IHC staining of T cells (CD3) and osteoblasts (Runx2, Col1a1) under the BCP implant treating with PBS or DT in vivo. Scale bar = 100 μm. Red arrow, positive cells. M, material. k–n Semiquantification of new bone area and positively stained cells in (i, j). n = 5, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001

Article Snippet: The primary antibodies were listed as: CD11c (1:200 for IF and 1:350 for IHC, Cat: 97,585, CST, USA), F4/80 (1:250, Cat: 70,076, CST, USA), Col1a1(1:100, Cat: SA2005, Boster, China), Runx2 (1:200, Cat: ab76956, Abcam, USA), CD146 (1:100, Cat: A13927, ABclonal, USA), CD83 (1:100, Cat: A2040, ABclonal, USA), CD40 (1:100, Cat: A0218, ABclonal, USA), iNOS (1:100, Sant Cruz, USA.), Arg1 (1:150, Cat: https://doi.org/10.1007/s40820-020-00540-z© The authors A11925, ABclonal, USA), CD3 (1:100, Cat: PB0112, Boster, China).

Techniques: Staining, In Vivo, Immunohistochemistry

Journal: eLife

Article Title: Biallelic mutations in calcium release activated channel regulator 2A (CRACR2A ) cause a primary immunodeficiency disorder

doi: 10.7554/eLife.72559

Figure Lengend Snippet: ( A ) A computerized tomography scan of the chest showing area of cylindrical bronchiectasis at the basal segment of the left lower lobe. ( B ) TCR repertoire as assessed by T receptor spectraphenotyping. Data are representative of one independent assay. ( C ) Phytohaemagglutinin (PHA) and ( D ) anti-CD3 T cell proliferation. CPM (counts per minute). Data are representative of three independent assays.

Article Snippet: Antibody , anti-human CD3 antibody (mouse monoclonal) , Bio X Cell , Clone OKT-3 , 1 μg/ml.

Techniques: Tomography

Journal: eLife

Article Title: Biallelic mutations in calcium release activated channel regulator 2A (CRACR2A ) cause a primary immunodeficiency disorder

doi: 10.7554/eLife.72559

Figure Lengend Snippet: Immunological characteristics of patient with variants in EFCAB4B.

Article Snippet: Antibody , anti-human CD3 antibody (mouse monoclonal) , Bio X Cell , Clone OKT-3 , 1 μg/ml.

Techniques:

Journal: eLife

Article Title: Biallelic mutations in calcium release activated channel regulator 2A (CRACR2A ) cause a primary immunodeficiency disorder

doi: 10.7554/eLife.72559

Figure Lengend Snippet: ( A ) T-helper (Th) 1, Th2 and Th17 cell distribution among CD4+ T cells in the peripheral blood. Grey bars healthy controls (N = 6), blue bars patient (two separate evaluations). ( B ) Representative dot plot shows the gating strategy. Whole EDTA blood was stained with a combination of CD3-V500, CD4-BV421, CCR6-Pe, and CXCR3-Alexa-Fluoro 647.

Article Snippet: Antibody , anti-human CD3 antibody (mouse monoclonal) , Bio X Cell , Clone OKT-3 , 1 μg/ml.

Techniques: Staining

Journal: eLife

Article Title: Biallelic mutations in calcium release activated channel regulator 2A (CRACR2A ) cause a primary immunodeficiency disorder

doi: 10.7554/eLife.72559

Figure Lengend Snippet: ( A ) Representative flow plots showing expression of IFN-γ in human PBMCs from a healthy donor and the patient. PBMCs were stimulated with anti-CD3 and anti-CD28 antibodies for 48 hr and cultured for further 4 days in the presence of IL-2 before re-stimulation with PMA plus ionomycin for 5 hr for cytokine analysis. Cells were gated for CD4+ T cells. ( B ) Quantitative mRNA expression analysis (± s.d.m.) of indicated cytokines from human PBMCs (cultured as mentioned above) with or without stimulation with PMA plus ionomycin for 5 hr. ( C ) Levels of IL-2 and TNF in human PBMCs from culture supernatants of cells stimulated as described above ( B ) were determined by ELISA. ( D ) Representative traces showing averaged (± SEM) SOCE responses from healthy control and patient PBMCs (cultured as indicated in A), after transient stimulation with anti-CD3 antibody cross-linking, or ionomycin (0.5 µM) in the presence of external solution containing 2 mM Ca 2+ (left) as indicated. Bar graphs show baseline subtracted ratio values for anti-CD3 antibody cross-linking or ionomycin (average± SEM) from six independent experiments (right). ( E ) Representative traces showing averaged (± SEM) SOCE responses from healthy control and patient PBMCs (cultured as indicated in A), after store-depletion with thapsigargin (1 µM) stimulation in Ca 2+ -free Ringer’s solution. SOCE was measured by addition of 2 mM Ca 2+ -containing Ringer’s solution as indicated (left). Bar graph shows baseline subtracted ratio values at the peak of SOCE (average± SEM) from four independent experiments (right). ( F ) Phosphorylated JNK levels in CD4+ T cells from healthy control and patient PBMCs (cultured as indicated in A), stimulated with anti-CD3 antibody for indicated times. Bar graphs show average± SEM from three independent experiments. ** p < 0.005, *** p < 0.0001.

Article Snippet: Antibody , anti-human CD3 antibody (mouse monoclonal) , Bio X Cell , Clone OKT-3 , 1 μg/ml.

Techniques: Expressing, Cell Culture, Enzyme-linked Immunosorbent Assay

Journal: eLife

Article Title: Biallelic mutations in calcium release activated channel regulator 2A (CRACR2A ) cause a primary immunodeficiency disorder

doi: 10.7554/eLife.72559

Figure Lengend Snippet: ( A ) Representative flow plots showing expression of IL-2 in control or CRACR2A KO Jurkat T cells stably expressing FLAG-tagged WT CRACR2A, CRACR2A E278D (E278D), or CRACR2A R144G/E300* (DM) after stimulation with PMA plus ionomycin for 16 hr (left). Bar graph shows means ± s.e.m. of pooled technical replicates from two independent experiments (right). ( B ) Representative flow plots showing expression of IFN-γ in primary human CD4+ T cells transduced with lentiviruses encoding CRACR2A -targeting sgRNA and those encoding cDNAs of WT or indicated mutants of CRACR2A after stimulation with anti-CD3 and anti-CD28 antibodies for 5 hr (left). Bar graph (right) shows means ± s.e.m. of pooled technical replicates from three independent experiments. ( C ) Representative flow plots showing expression of IFN-γ in primary human CD4+ T cells purified from healthy donors and transduced with lentiviruses encoding cDNAs for WT or indicated mutant of CRACR2A after stimulation with anti-CD3 and anti-CD28 antibodies for 5 hr (left). Bar graph (right) shows means ± s.e.m. of pooled technical replicates from three independent experiments. * p < 0.05, *** p < 0.0001.

Article Snippet: Antibody , anti-human CD3 antibody (mouse monoclonal) , Bio X Cell , Clone OKT-3 , 1 μg/ml.

Techniques: Expressing, Stable Transfection, Transduction, Purification, Mutagenesis

Journal: eLife

Article Title: Biallelic mutations in calcium release activated channel regulator 2A (CRACR2A ) cause a primary immunodeficiency disorder

doi: 10.7554/eLife.72559

Figure Lengend Snippet: ( A ) Representative traces showing averaged SOCE from control (48 cells) or CRACR2A-KO Jurkat T cells (KO) transduced with an empty vector (55 cells) or those encoding FLAG-tagged WT CRACR2A (45 cells), CRACR2A E278D (E278D, 40 cells), or CRACR2A R144G/E300* (DM, 50 cells) mutants (left). Cells were stimulated with anti-CD3 antibodies, followed by ionomycin treatment in the presence of external solution containing 2 mM Ca 2+ . Bar graphs show averaged baseline subtracted peak SOCE (± s.e.m.) from anti-CD3 antibody and ionomycin treatments, from three independent experiments (right). ( B ) Representative traces showing averaged SOCE induced by thapsigargin (TG) treatment from control (52 cells) or CRACR2A-KO Jurkat T cells (KO) transduced with empty vector (46 cells) or those encoding FLAG-tagged WT CRACR2A (49 cells), CRACR2A E278D (E278D, 51 cells), or CRACR2A R144G/E300* (DM, 50 cells) mutants (top). Cells were stimulated with thapsigargin in Ca 2+ -free solution to deplete the intracellular stores and exposed to external solution containing 2.0 mM Ca 2+ . Bar graphs below show averaged baseline subtracted SOCE levels (± s.e.m.) at the peak (center) or later time point (sustained – 900 s, bottom) from three independent experiments. ( C ) Phosphorylated JNK levels in control or CRACR2A KO Jurkat T cells stably expressing WT and indicated mutants of CRACR2A, stimulated with anti-CD3 antibody for indicated times. Graphs show average± SDM from three independent experiments. * p < 0.05, ** p < 0.005, *** p < 0.0001.

Article Snippet: Antibody , anti-human CD3 antibody (mouse monoclonal) , Bio X Cell , Clone OKT-3 , 1 μg/ml.

Techniques: Transduction, Plasmid Preparation, Stable Transfection, Expressing

Journal: eLife

Article Title: Biallelic mutations in calcium release activated channel regulator 2A (CRACR2A ) cause a primary immunodeficiency disorder

doi: 10.7554/eLife.72559

Figure Lengend Snippet: ( A ) Immunoprecipitation for detection of binding between WT and indicated mutants of CRACR2A (E278D, or DM) with ORAI1/STIM1. Lysates of HEK293T cells expressing FLAG-tagged CRACR2A in the presence of 6 X His-tagged ORAI1 and STIM1, were subjected to immunoprecipitation with anti-FLAG antibodies and analyzed by immunoblotting for detection of the indicated proteins. Bar graphs below show densitometry analysis of binding of CRACR2A to ORAI1 (left) and STIM1 (right), normalized to that of WT CRACR2A, from three independent experiments. ( B ) Immunoprecipitation for detection of binding between CRACR2A and VAV1. Lysates of HEK293T cells expressing FLAG-tagged WT or indicated mutants of CRACR2A in the presence of GFP-tagged VAV1, were subjected to immunoprecipitation with anti-FLAG antibodies and analyzed by immunoblotting for detection of indicated proteins. Bar graph below shows densitometry analysis of binding of CRACR2A to VAV1, normalized to that of WT CRACR2A, from three independent experiments. ( C ) Representative confocal images of CRACR2A KO Jurkat cells stably expressing N-terminally FLAG-tagged WT or indicated mutants of CRACR2A under resting conditions (top panels) or 20 min after dropping on stimulatory anti-CD3 antibody-coated coverslips (middle and bottom panels). The top panels showed images from the center of the cell. The middle panels show images from the bottom of the cell, which was in contact with the coverslip and the bottom panels showed images from the center of the anti-CD3 antibody-stimulated cell. F-Actin staining – green, anti-FLAG antibody staining – red. Images are representative of at least 10 cells in each condition. * p < 0.05, ** p < 0.005.

Article Snippet: Antibody , anti-human CD3 antibody (mouse monoclonal) , Bio X Cell , Clone OKT-3 , 1 μg/ml.

Techniques: Immunoprecipitation, Binding Assay, Expressing, Western Blot, Stable Transfection, Staining

Journal: eLife

Article Title: Biallelic mutations in calcium release activated channel regulator 2A (CRACR2A ) cause a primary immunodeficiency disorder

doi: 10.7554/eLife.72559

Figure Lengend Snippet:

Article Snippet: Antibody , anti-human CD3 antibody (mouse monoclonal) , Bio X Cell , Clone OKT-3 , 1 μg/ml.

Techniques: Irradiation, Transfection, Construct, Plasmid Preparation, Isolation, Flow Cytometry, Recombinant, Sequencing, Cell Isolation, Enzyme-linked Immunosorbent Assay, Quantitation Assay, Staining, Software, Imaging, Microscopy