myd88 mutant gyrb cdna  (Addgene inc)

Journal: eBioMedicine

Article Title: Cytotoxic CD161 − CD8 + T EMRA cells contribute to the pathogenesis of systemic lupus erythematosus

doi: 10.1016/j.ebiom.2023.104507

Figure Lengend Snippet: DTHD1 deficiency increased the cytotoxicity of CD161 − CD8 + T EMRA cells . (a) Heatmap showing the correlation of co-expression modules with different cell subsets. (b) Functional annotations of DTHD1-related genes. (c) Quantification (left) and flow cytometry (right) of the apoptosis percentage of P815 cells after cocultured with primary CD8 + T cells electroporated with si-NC or si- DTHD1 siRNAs for 5 h at the ratio of 1:5. P value is determined by Unpaired t-test, P = 0.0124. (d) Flow cytometric analysis of mean fluorescence intensity (MFI) of Granzyme B of CD161 − CD8 + T cells electroporated with si-NC or si- DTHD1 siRNAs after coculture with P815 cell. P value is calculated by Unpaired t-test, P = 0.0012. (e) Structural prediction of DTHD1-MYD88 interaction by ZDOCK. Grey, DTHD1; blue, MYD88; red, mutated regions in DTHD1 . (f) Western blotting analysis of HA in HEK293T cells co-transfected with myc-MYD88 and HA-empty vector or HA-DTHD1 (WT) or HA-DTHD1 (Mutant) expressing plasmids after immunoprecipitated with anti-Myc beads. (g) Boxplot indicating the average expression of MYD88 in CD161 − CD8 + T EMRA cells in HCs and SLE samples from our dataset. P value is from Wilcoxon rank-sum test. (h) Luciferase activity analysis of lysates of HEK293T cells co-transfected luciferase reporter plasmid for NF-κB, pRL-TK-renilla-luciferase plasmid, MYD88 plasmid and WT-DTHD1 plasmid or mutant DTHD1 plasmid ( n = 6). P values are determined by Unpaired t-test. (i) Quantification (left) and flow cytometry (right) of the apoptosis percentage of P815 cells after cocultured with primary CD8 + T cells pre-treated with DMSO or TAK-242 for 5 h at the ratio of 1:5. P value is determined by Unpaired t-test, P = 0.0024. One representative experiment of three is shown (f). ∗ P < 0.05, ∗∗ P < 0.01, ∗∗∗ P < 0.001.

Article Snippet: Human MYD88 cDNA was purchased from Genecopoeia Inc. and cloned into the pcDNA3.1-Myc plasmid.

Techniques: Expressing, Functional Assay, Flow Cytometry, Fluorescence, Structural Proteomics, Western Blot, Transfection, Plasmid Preparation, Mutagenesis, Immunoprecipitation, Luciferase, Activity Assay

Journal: eBioMedicine

Article Title: Cytotoxic CD161 − CD8 + T EMRA cells contribute to the pathogenesis of systemic lupus erythematosus

doi: 10.1016/j.ebiom.2023.104507

Figure Lengend Snippet: CD161 − CD8 + T EMRA cells contribute to SLE by LIGHT signaling . (a) The number (top) and strength (bottom) of interaction among all cells in HCs and SLE samples. (b) Heatmap of differential interactions between HCs and SLE samples in cell–cell communication network. The top bar indicates the sum of incoming signaling and right bar indicates the sum of outgoing signaling. Red indicates increased signaling and blue indicates decreased signaling in SLE. (c) Number of significant ligand-receptor pairs between CD161 − CD8 + T EMRA cells (outgoing) and other cell subclusters (incoming) in HCs (left) and SLEs (right). The relative number of ligand-receptor pairs is represented by the edge width. (d) Comparison of the significant outgoing signaling from CD161 − CD8 + T EMRA cells between HC and SLE. Empty space means the communication probability is zero. P -values are computed from one-sided permutation test. ∗∗∗ P < 0.001. (e) qPCR analysis of TNFSF14 expression in primary CD8 + T cells electroporated with empty vector, DTHD1-WT or DTHD-mut plasmid cocultured with THP1 cells ( n = 3). P values are determined by unpaired t-test. (f) Receiver operating curve for out-of-sample prediction of case–control state by a logistic regression model trained on DEGs in CD161 − CD8 + T EMRA cells. DEGs include IFI27 , IFI44L , RSAD2 , IFI44 , FAM118A , LGALS9 , MX1 , EPSTI1 , USP18 , OAS3 , LAIR2 , IFIT1 , XAF1 . Inset depicts the changes of DEGs in the public transcriptome profile and CD161 − CD8 + T EMRA cells. (g) Graphic abstract showing the expansion of CD161 − CD8 + T EMRA in patients with SLE and DTHD1 downregulation promotes MYD88-mediated expansion and cytotoxicity of this pathogenic CD161 − CD8 + T EMRA subset in SLE.

Article Snippet: Human MYD88 cDNA was purchased from Genecopoeia Inc. and cloned into the pcDNA3.1-Myc plasmid.

Techniques: Comparison, Expressing, Plasmid Preparation, Control

Journal: Molecular cell

Article Title: OTUD4 is a phospho-activated K63 deubiquitinase that regulates MyD88-dependent signaling

doi: 10.1016/j.molcel.2018.01.009

Figure Lengend Snippet: (a) Flag immunoprecipitation was performed from HEK293 cells expressing the indicated vectors and Western blotted as shown. Results are representative of three independent experiments. (b) HA immunoprecipitation was performed after expression of the indicated vectors and analyzed as shown. Results are representative of two independent experiments. (c) Whole cell extracts from mouse embryonic fibroblasts (MEF WCE) was immunoprecipitated using control IgG or OTUD4 antibody and Western blotted as shown. Results are representative of three independent experiments. (d) MBP and MBP-OTUD4 were co-expressed in E. coli with His-Flag-MyD88. After MBP pulldown, the bound material was analyzed by Western blot using the indicated antibodies. Results are representative of three independent experiments. (e) Flag-MyD88 and HA-Ub WT or mutants were expressed in HEK293 cells, Flag-immunoprecipitated after SDS denaturation, and blotted as shown. Results are representative of two independent experiments. (f) Flag immunoprecipitation was performed after SDS denaturation from HEK293 cells expressing Flag-MyD88 and HA-Ub (WT), along with OTUD4WT, OTUD4C45A, or empty vector as indicated. (g) K63-linked (K63 only) ubiquitination status of MyD88 was assessed as in (f). Results for (f) and (g) are representative of two independent experiments for each. Flag immunoprecipitation was performed from HEK293 cells expressing HA-tagged WT ubiquitin (h) or K63-linked ubiquitin (i), along with vectors as indicated. Results for (h) and (i) are representative of two independent experiments for each.

Article Snippet: MyD88 cDNA was obtained from Addgene (clone #12287), cloned by PCR into pENTR-3C and subcloned into the expression vectors as above.

Techniques: Immunoprecipitation, Expressing, Western Blot, Control, Plasmid Preparation, Ubiquitin Proteomics

Journal: Molecular cell

Article Title: OTUD4 is a phospho-activated K63 deubiquitinase that regulates MyD88-dependent signaling

doi: 10.1016/j.molcel.2018.01.009

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: MyD88 cDNA was obtained from Addgene (clone #12287), cloned by PCR into pENTR-3C and subcloned into the expression vectors as above.

Techniques: Western Blot, Microscopy, Ubiquitin Proteomics, Purification, Luciferase, Enzyme-linked Immunosorbent Assay, Recombinant, Construct, shRNA