Journal: The Journal of Clinical Investigation

Article Title: Anti-CD45RC antibody immunotherapy prevents and treats experimental autoimmune polyendocrinopathy–candidiasis–ectodermal dystrophy syndrome

doi: 10.1172/JCI156507

Figure Lengend Snippet: ( A ) Schematic of the protocol of administration of isotype control or anti-CD45RC mAbs as prevention in 3-week-old Aire –/– rats. ( B ) Representative photographs of Aire –/– rats after 4 months of treatment with either isotype control ( n = 13) or anti-CD45RC mAbs ( n = 13). Arrows indicate the alopecia- and vitiligo-like manifestations. Scale bars: 5 cm. ( C ) Evolution of weight gain in Aire –/– male rats during treatment with isotype control ( n = 6) or anti-CD45RC mAbs ( n = 6) until sacrifice. ANOVA comparing curves: *** P < 0.001. ( D ) Representative pictures of H&E histological analysis of organs from Aire –/– rats at the end of the 4-month treatment with isotype control ( n = 7) or anti-CD45RC mAbs ( n = 7). Black arrows indicate lesions and mononuclear cell infiltrates. Black bars indicate the marginal zone. Scale bars: 250 nm (thymus and spleen), 500 μm (skin and kidney), and 1 mm (pancreas and lung).

Article Snippet: The slides were immersed in 3% Tris–goat serum solution to block nonspecific binding, and then 1:200 CD45RC (clone MT2, catalog AM39022PU-N, Origene) antibody was applied at room temperature and incubated for 1 hour.

Techniques:

Journal: The Journal of Clinical Investigation

Article Title: Anti-CD45RC antibody immunotherapy prevents and treats experimental autoimmune polyendocrinopathy–candidiasis–ectodermal dystrophy syndrome

doi: 10.1172/JCI156507

Figure Lengend Snippet: ( A ) Tissue sections of organs from IgM –/– rats were incubated with sera of Aire –/– animals at 4 months of treatment with anti-CD45RC or isotype control mAb. Autoantibodies are depicted in yellow and DAPI in blue. Original magnification, ×20. Images are representative of 3 different experiments. ( B ) Sera from anti-CD45RC or isotype control mAb–treated Aire –/– rats at 4 months of treatment were incubated on Western blot membranes after migration and transfer of tissue-specific self-antigens from IgM –/– rats. Binding of autoantibodies was revealed using an anti-rat IgG biotin-coupled antibody and avidin peroxidase. β-Actin was used as a loading control. Data are representative of 5 different experiments. MLN, mesenteric lymph nodes. ( C ) Anti-cytokine and tissue-specific autoantibodies were quantified by LIPS assay using sera from anti-CD45RC or isotype control mAb–treated Aire –/– rats at 4 months of treatment. Normalization was achieved by division of the obtained value by the mean values obtained from WT animals. Mann-Whitney analysis showed no significant difference between the 2 groups.

Article Snippet: The slides were immersed in 3% Tris–goat serum solution to block nonspecific binding, and then 1:200 CD45RC (clone MT2, catalog AM39022PU-N, Origene) antibody was applied at room temperature and incubated for 1 hour.

Techniques: Incubation, Western Blot, Migration, Binding Assay, Avidin-Biotin Assay, Lips Assay, MANN-WHITNEY

Journal: The Journal of Clinical Investigation

Article Title: Anti-CD45RC antibody immunotherapy prevents and treats experimental autoimmune polyendocrinopathy–candidiasis–ectodermal dystrophy syndrome

doi: 10.1172/JCI156507

Figure Lengend Snippet: ( A – C ) Blood at 2 weeks of treatment ( A ) and spleen ( B ) and thymus ( C ) at 4 months of treatment of Aire –/– rats with isotype control or anti-CD45RC mAbs were stained for the expression of CD45RC on CD8 + and CD4 + T cells by flow cytometry and compared with those from Aire +/+ rats. ( A ) Shown is a representative staining of 4–7 animals. The gates indicate the high, low, and negative subsets of CD45RC. Mean ± SEM of CD45RC expression on CD4 + and CD8 + T cells after 2 weeks of treatment is summarized in the graphs on the right. ANOVA: * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001. ( B and C ) Results are shown as mean ± SEM of CD45RC subsets among CD4 + T cells (left) or CD8 + T cells (right). ANOVA: * P < 0.05, ** P < 0.01, *** P < 0.001. ( D ) Cell subset distribution was analyzed by flow cytometry among immune cells from spleen, mesenteric lymph nodes (MLN), and thymus of untreated Aire +/+ and isotype control or anti-CD45RC mAb–treated Aire –/– rats at the time of sacrifice. Results are shown as mean ± SEM. ANOVA: *** P < 0.001, **** P < 0.0001.

Article Snippet: The slides were immersed in 3% Tris–goat serum solution to block nonspecific binding, and then 1:200 CD45RC (clone MT2, catalog AM39022PU-N, Origene) antibody was applied at room temperature and incubated for 1 hour.

Techniques: Staining, Expressing, Flow Cytometry

Journal: The Journal of Clinical Investigation

Article Title: Anti-CD45RC antibody immunotherapy prevents and treats experimental autoimmune polyendocrinopathy–candidiasis–ectodermal dystrophy syndrome

doi: 10.1172/JCI156507

Figure Lengend Snippet: ( A ) Proportion of B cell subtypes in the spleen of Aire –/– rats after 4 months of treatment with the anti-CD45RC or isotype control mAb or WT untreated rats. ( B ) B cells from Aire +/+ rats were stimulated in vitro for 48 hours with CpG ODN 1668, anti-CD40, and anti-IgM mAbs in the presence of anti-CD45RC or isotype control mAbs. Maturation of B cells was analyzed by flow cytometry quantification of each B cell subpopulation. ANOVA: * P < 0.05. ( C ) After 48 hours of in vitro stimulation, B cells from Aire +/+ and Aire –/– rats were stained for flow cytometry to study their viability and expression of CD45RC and activation markers such as CD80, CD40, and MHC class II (RT1-B). Data from each experiment were normalized to the mean value from all the experiments. Results of multiple t test statistical analysis comparing isotype control with anti-CD45RC mAb conditions in Aire –/– or Aire +/+ B cells. Multiple t test: * P < 0.05, ** P < 0.01, *** P < 0.001. ( D ) IgM and IgG ELISA quantification in the culture supernatant of B cells stimulated for 48 hours. Multiple t test: * P < 0.05. ( E ) Ratio of IgM versus IgG production in culture supernatant of B cells stimulated for 48 hours.

Article Snippet: The slides were immersed in 3% Tris–goat serum solution to block nonspecific binding, and then 1:200 CD45RC (clone MT2, catalog AM39022PU-N, Origene) antibody was applied at room temperature and incubated for 1 hour.

Techniques: In Vitro, Flow Cytometry, Staining, Expressing, Activation Assay, Enzyme-linked Immunosorbent Assay

Journal: The Journal of Clinical Investigation

Article Title: Anti-CD45RC antibody immunotherapy prevents and treats experimental autoimmune polyendocrinopathy–candidiasis–ectodermal dystrophy syndrome

doi: 10.1172/JCI156507

Figure Lengend Snippet: ( A ) Ratio of CD4 + CD25 + CD127 lo/– or CD8 + CD45RC lo/– Tregs versus CD45RC hi Tconv cells in Aire –/– rats treated with isotype control ( n = 7) versus anti-CD45RC mAb ( n = 7). ANOVA: * P < 0.05. ( B ) Matrix showing the number of genes differentially expressed between CD4 + (left panel) and CD8 + (right panel) Tregs from the following groups: WT rats ( n = 8) and Aire –/– rats treated with the isotype control ( n = 5) or the anti-CD45RC mAb ( n = 5). ( C ) DGE RNA sequencing heatmap analysis of CD8 + CD45RC lo Tregs showing the relative expression of genes. Columns correspond to samples, and rows correspond to differentially expressed genes. Expression values were averaged per sample and scaled per gene. Blue represents lowly-expressed genes and red represents highly expressed genes. ( D ) Normalized enrichment score of biological pathways upregulated or downregulated in Aire –/– rats treated with the anti-CD45RC mAb ( n = 5) compared with Aire –/– rats treated with the isotype control mAb ( n = 5).

Article Snippet: The slides were immersed in 3% Tris–goat serum solution to block nonspecific binding, and then 1:200 CD45RC (clone MT2, catalog AM39022PU-N, Origene) antibody was applied at room temperature and incubated for 1 hour.

Techniques: RNA Sequencing Assay, Expressing

Journal: The Journal of Clinical Investigation

Article Title: Anti-CD45RC antibody immunotherapy prevents and treats experimental autoimmune polyendocrinopathy–candidiasis–ectodermal dystrophy syndrome

doi: 10.1172/JCI156507

Figure Lengend Snippet: ( A ) Schematic of the protocol of treatment in the curative setting of 3-month-old Aire –/– rats with isotype control or anti-CD45RC mAbs. ( B ) Representative photographs of Aire –/– rats after 4 months of treatment with either isotype control ( n = 8) or anti-CD45RC mAbs ( n = 11). Arrows depict alopecia. Scale bars: 5 cm. ( C ) Evolution of weight gain in male Aire –/– rats treated with isotype control ( n = 4) or anti-CD45RC mAbs ( n = 5) until sacrifice. ANOVA analysis showed no significant difference between the 2 groups. ( D ) Representative pictures of H&E histological analysis of organs from Aire –/– rats at the end of the treatment with isotype control ( n = 4) or anti-CD45RC mAbs ( n = 5). Black arrows indicate autoimmune lesions and immune cell infiltrates. Black bars indicate the marginal zone. Scale bars: 250 nm (spleen, lung, and kidney), 500 μm (skin and pancreas), and 1 mm (thymus).

Article Snippet: The slides were immersed in 3% Tris–goat serum solution to block nonspecific binding, and then 1:200 CD45RC (clone MT2, catalog AM39022PU-N, Origene) antibody was applied at room temperature and incubated for 1 hour.

Techniques:

Journal: The Journal of Clinical Investigation

Article Title: Anti-CD45RC antibody immunotherapy prevents and treats experimental autoimmune polyendocrinopathy–candidiasis–ectodermal dystrophy syndrome

doi: 10.1172/JCI156507

Figure Lengend Snippet: ( A ) Organ serial sections from the same IgM –/– rats as shown in were incubated with different sera from anti-CD45RC or isotype mAb–treated Aire –/– animals at 4 months of treatment as indicated. Autoantibodies are depicted in yellow and DAPI in blue. Original magnification, ×20. Images are representative of 3 animals per group. ( B ) Sera from anti-CD45RC or isotype mAb–treated Aire –/– rats at 4 months of treatment were incubated on Western blot membranes after migration and transfer of tissue-specific self-antigens from IgM –/– rats. β-Actin was used as a loading control. Data are representative of 5 animals per group. MLN, mesenteric lymph nodes. ( C ) Anti-cytokine and -Pdia2 autoantibodies were quantified by LIPS assay using sera from anti-CD45RC or isotype mAb–treated Aire –/– rats at 4 months of treatment ( n = 5 each). Normalization was achieved by division of the obtained value by the mean values obtained from WT animals. Mann-Whitney: * P < 0.05, ** P < 0.01.

Article Snippet: The slides were immersed in 3% Tris–goat serum solution to block nonspecific binding, and then 1:200 CD45RC (clone MT2, catalog AM39022PU-N, Origene) antibody was applied at room temperature and incubated for 1 hour.

Techniques: Incubation, Western Blot, Migration, Lips Assay, MANN-WHITNEY

Journal: The Journal of Clinical Investigation

Article Title: Anti-CD45RC antibody immunotherapy prevents and treats experimental autoimmune polyendocrinopathy–candidiasis–ectodermal dystrophy syndrome

doi: 10.1172/JCI156507

Figure Lengend Snippet: ( A ) PBMCs of APECED patients ( n = 11) and healthy donors ( n = 16) were stained for flow cytometry analysis showing the expression of CD45RC on CD4 + (left) and CD8 + (right) T cells; t test: * P < 0.05, ** P < 0.01, *** P < 0.001. ( B ) Expression of FOXP3 and CD45RC on CD4 + (top line) and CD8 + T cells (bottom line) from APECED patients and healthy donors. ( C ) Ratio of FOXP3 + Tregs versus CD45RC hi Tconv cells in healthy donors ( n = 16) versus APECED patients ( n = 11); t test: * P < 0.05. ( D ) Expression of IL-10, IL-34, CD40L, Tbet, CD103, CD127, and PD-1 by CD4 + and CD8 + T cells from healthy donors and APECED patients; t test: * P < 0.05, ** P < 0.01, *** P < 0.001. ( E ) Representative immunohistochemical staining of CD45RC with an anti–human CD45RC mAb in stomach and small intestine paraffin-embedded tissue from 2 APECED patients with autoimmune gastritis and enteropathy (arrows) compared with stomach tissue of an APECED patient without autoimmune gastritis. Non-APECED human tonsil biopsy tissue was used as positive control. ( F ) Proportion of apoptotic CD45RA hi T cells induced after a 3-hour in vitro incubation of PBMCs, from healthy donors ( n = 6) or APECED patients ( n = 6) with the anti-CD45RC or isotype control mAbs. One-way ANOVA repeated measures, Bonferroni’s post hoc test: *** P < 0.001, **** P < 0.0001.

Article Snippet: The slides were immersed in 3% Tris–goat serum solution to block nonspecific binding, and then 1:200 CD45RC (clone MT2, catalog AM39022PU-N, Origene) antibody was applied at room temperature and incubated for 1 hour.

Techniques: Staining, Flow Cytometry, Expressing, Immunohistochemical staining, Positive Control, In Vitro, Incubation

Journal: PLoS ONE

Article Title: CD45RC Isoform Expression Identifies Functionally Distinct T Cell Subsets Differentially Distributed between Healthy Individuals and AAV Patients

doi: 10.1371/journal.pone.0005287

Figure Lengend Snippet: Peripheral blood leukocytes from 39 healthy individuals (median age 55, range 25–70) were stained with mAbs against CD3, CD4, CD8, CD45RC. (A) The histograms represent the CD45RC expression on CD4 T cells (left panel) and CD8 T cells (right panel) from two healthy individuals showing the inter-individual variability in CD45RC expression. (B) The proportion of CD45RC low CD4 T cells (left panel) and the proportion of CD45RC high -CD45RC int -CD45RC low CD8 T cells (right panels) are presented according to age of the donors. Each dot represents a separate individual. The r- and p-values were calculated using linear regression. (C) Represent the percentage of CD45RC low CD4 T cells (left panel) or the proportion of CD8 CD45RC T cell subsets (right panel) of 11 individuals at 4 years interval. The p-values were calculated using the Wilcoxon matched-pairs test; *, p<0.05.

Article Snippet: The isolation of CD45RC high and CD45RC low CD4 T cell subsets was performed as follows: CD4 T cells were stained with limiting amounts of FITC-conjugated anti-CD45RC mAb and separated into CD45RC high and CD45RC low cells by positive selection after addition of anti-FITC MACS microbeads (Miltenyi).

Techniques: Staining, Expressing

Journal: PLoS ONE

Article Title: CD45RC Isoform Expression Identifies Functionally Distinct T Cell Subsets Differentially Distributed between Healthy Individuals and AAV Patients

doi: 10.1371/journal.pone.0005287

Figure Lengend Snippet: Peripheral blood leukocytes from healthy individuals were stained with mAbs against TCR, CD4 or CD8, CD45RC, CD45RA, CD45RO and CCR7 (n = 6) or TCR, CD45RC, CD4 or CD8 and Foxp3 (n = 27). Gates were set on CD4 T cells (upper panels) or CD8 T cells (lower panels). Box plot diagrams represent the proportion of naive (CD45RA+CD45RO−CCR7+), central memory (CD45RA−CD45RO+CCR7+), effector memory (CD45RA−CD45RO+CCR7−) and natural regulatory T cells (Foxp3+) within the CD45RC subsets. The group “others” contains both CD45RA+CD45RO−CCR7− and CD45RA+CD45RO+ subsets, subpopulations with ill defined functions. The p-values were calculated using the Wilcoxon matched-pairs test; *, p<0.05; **, p<0.02; p<0.002.

Article Snippet: The isolation of CD45RC high and CD45RC low CD4 T cell subsets was performed as follows: CD4 T cells were stained with limiting amounts of FITC-conjugated anti-CD45RC mAb and separated into CD45RC high and CD45RC low cells by positive selection after addition of anti-FITC MACS microbeads (Miltenyi).

Techniques: Staining

Journal: PLoS ONE

Article Title: CD45RC Isoform Expression Identifies Functionally Distinct T Cell Subsets Differentially Distributed between Healthy Individuals and AAV Patients

doi: 10.1371/journal.pone.0005287

Figure Lengend Snippet: Peripheral blood leukocytes from 39 healthy individuals (HC), 38 patients with ANCA-associated vasculitis (AAV), and 20 patients with systemic lupus erythematosus (SLE), were stained with mAbs against CD3, CD4, CD8, CD45RC. (A) The proportion of CD45RC low CD4 T cells (left panel) and the proportion of CD45RC high -CD45RC int -CD45RC low CD8 T cells (right three panels) are presented as box plot diagrams for each study population. The p-values were calculated using the Wilcoxon matched-pairs test; p<0.05; **, p<0.02; ***, p<0.002. (B) The proportion of CD45RC low CD4 T cells are presented according to disease subtype (WG, Wegener's granulomatosis; MPA, microscopic polyangiitis; CSS, Churg-Strauss Syndrome; RLV, renal limited vasculitis), type of ANCA specificity (MPO, myeloperoxidase; PR3, proteinase 3), renal involvement (no: no kidney disease; yes: kidney disease), and relapses (no: no relapse; yes: relapses). Data are presented as box plot diagrams for each study population. The p-values were calculated using Mann Witney U test; *p<0.05. The proportion of CD45RC low CD4 T cells are presented according to duration of disease (C, left panel). The proportion of CD45RC low CD4 T cells of 18 AAV patients (13 WG, 3 MPA, and 2 RLV patients) at 4 years interval (C, right panel).

Article Snippet: The isolation of CD45RC high and CD45RC low CD4 T cell subsets was performed as follows: CD4 T cells were stained with limiting amounts of FITC-conjugated anti-CD45RC mAb and separated into CD45RC high and CD45RC low cells by positive selection after addition of anti-FITC MACS microbeads (Miltenyi).

Techniques: Staining

Journal: PLoS ONE

Article Title: CD45RC Isoform Expression Identifies Functionally Distinct T Cell Subsets Differentially Distributed between Healthy Individuals and AAV Patients

doi: 10.1371/journal.pone.0005287

Figure Lengend Snippet: (A) Representative example of the purification of CD4 CD45RC T cell subsets. Results are shown as histograms for CD45RC expression on CD4 T cells before (left histogram) and after CD45RC subsets purification (right histograms). The values within the histograms represent the percentage of CD45RC T cell subsets. (B) Purified CD45RC high (High) and CD45RC low (Low) CD4 T cell subsets, were stimulated in vitro with plate-bound anti-CD3 and anti-CD28 mAbs. The supernatants were collected at 72 h of culture and analyzed for the presence of cytokines using the CBA kit and Elisa. The results obtained in 20 healthy individuals are presented as box plot diagrams. The p-values were calculated using the Wilcoxon matched-pairs test; *, p<0.05; **, p<0.02; ***, p<0.002. (C) For intracellular measurement of cytokines, purified CD4 CD45RC high and CD45RC low T cells were stimulated and stained using FITC-labeled anti-IFN-γ mAb and PE-labeled anti-IL-4 or anti-IL-10 mAbs. The results are expressed as dot plot representing IFN-γ/IL-4 or IFN-γ/IL-10 production by CD4 T cell subsets. The values within the plots represent the fraction of CD4 T cells producing the indicated cytokine. The results are representative of three independent experiments.

Article Snippet: The isolation of CD45RC high and CD45RC low CD4 T cell subsets was performed as follows: CD4 T cells were stained with limiting amounts of FITC-conjugated anti-CD45RC mAb and separated into CD45RC high and CD45RC low cells by positive selection after addition of anti-FITC MACS microbeads (Miltenyi).

Techniques: Purification, Expressing, In Vitro, Enzyme-linked Immunosorbent Assay, Staining, Labeling

Journal: PLoS ONE

Article Title: CD45RC Isoform Expression Identifies Functionally Distinct T Cell Subsets Differentially Distributed between Healthy Individuals and AAV Patients

doi: 10.1371/journal.pone.0005287

Figure Lengend Snippet: (A) Representative example of the purification of CD45RC high , CD45RC int and CD45RC low CD8 T cell subsets. Results are shown as histograms for CD45RC expression on CD8 T cells before (left histogram) and after CD45RC subsets purification (right histograms). The values within the histograms represent the percentage of CD45RC T cell subsets. (B) These sub-populations were stimulated in vitro with anti-CD3 and anti-CD28 mAbs. The supernatants were collected at 96 h of culture and analyzed for the presence of cytokines using the CBA kit and Elisa. The results obtained in 12 healthy individuals are presented as box plot diagrams. The p-values were calculated using the Wilcoxon matched-pairs test; **, p<0.02. (C) For intracellular measurement of cytokines, purified CD8 CD45RC T cell subsets were stimulated and analyzed for intracytoplasmic cytokines as indicated in the legend of . The results are representative of three independent experiments.

Article Snippet: The isolation of CD45RC high and CD45RC low CD4 T cell subsets was performed as follows: CD4 T cells were stained with limiting amounts of FITC-conjugated anti-CD45RC mAb and separated into CD45RC high and CD45RC low cells by positive selection after addition of anti-FITC MACS microbeads (Miltenyi).

Techniques: Purification, Expressing, In Vitro, Enzyme-linked Immunosorbent Assay

Journal: The Journal of Biological Chemistry

Article Title: Cytoplasmic poly(A)-binding protein 1 (PABPC1) interacts with the RNA-binding protein hnRNPLL and thereby regulates immunoglobulin secretion in plasma cells

doi: 10.1074/jbc.M117.794834

Figure Lengend Snippet: PABPC1 is not required for CD45 alternative splicing. A, Jurkat cells were transduced with PLKO.1 lentivirus containing either shPabpc1 or a Scrambled sequence, and expression of PABPC1 was determined by immunoblotting analysis. GAPDH was served as a loading control. B, PABPC1 was not required for activation-induced CD45 isoforms switching in Jurkat cells. As in A, Jurkat cells stably expressing the shRNAs against PABPC1 or Scramble were stimulated with PMA, and expression of CD45RA and CD45RO was determined by flow cytometry. C, naïve B cells were isolated from C57BL/6 mice and stimulated with 10 μg/ml LPS in the presence of IL-4 (10 ng/ml) and IL-5 (10 ng/ml). The activated B cells were then transduced with retrovirus containing three independent shRNAs against Pabpc1 (shPabpc1) or a scrambled sequence (shCtrl) at 16 and 40 h after stimulation, and 1 μg/ml puromycin was added into cultured cells at 48 h after stimulation. At 96 h after stimulation, the expression of PABPC1 was analyzed by qRT-PCR. D and E, PABPC1 was not required for CD45 splicing in activated B cells. As in C, RNA was extracted from unstimulated B cells, LPS-stimulated B cells (mock), LPS-stimulated B cells transduced with retrovirus containing a scrambled sequence (shCtrl), or three independent shRNAs against Pabpc1 (shPabpc1). CD45 splicing was analyzed by RT-PCR; PCR products corresponding to each splicing isoform of CD45 are indicated (D); and cell surface expression of CD45RA, CD45RB, CD45RC, and B220 (CD45RABC) was determined by flow cytometry (E). E, red indicates the cells transduced with shCtrl, and green, blue, and cyan indicate the cells transduced with one of the shPabpc1 shRNAs. F and G, hnRNPLL was required for exclusion of CD45RA exon. Jurkat cells were stably transduced with shRNA against either hnRNPLL (shLL) or a Scrambled sequence (shCtrl). Expression of hnRNPLL was determined by immunoblotting (F), and expression of CD45RA and CD45RO was determined by flow cytometry (G). H and I, the short but not the long isoform of hnRNPLL regulates CD45 splicing. A20 cells were transduced with control construct (mock) or long or short isoform of hnRNPLL (LL-long or LL-short), respectively, and expression of the hnRNPLL was analyzed by immunoblotting (H). CD45 splicing was analyzed by RT-PCR, and the PCR products corresponding to each splicing isoform of CD45 are indicated (I). The data are representative of at least three independent experiments.

Article Snippet: Flow cytometry Primary B cells were activated and transduced shRNA retrovirus as described above and were stained with PE anti-mouse CD45RA (BD Pharmingen), PE anti-mouse CD45RC (BD Pharmingen), Alexa Fluro 647 anti-mouse CD45RC (Biolegend), and APC anti-mouse CD45R/B220 (Biolegend) at 96 h after activation.

Techniques: Transduction, Sequencing, Expressing, Western Blot, Activation Assay, Stable Transfection, Flow Cytometry, Isolation, Cell Culture, Quantitative RT-PCR, Reverse Transcription Polymerase Chain Reaction, shRNA, Construct