Journal: Immunity

Article Title: m 6 A Modification Prevents Formation of Endogenous Double-Stranded RNAs and Deleterious Innate Immune Responses during Hematopoietic Development

doi: 10.1016/j.immuni.2020.05.003

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Cy3 Streptavidin , VECTOR LABORATORIES , Cat# SA-1300.

Techniques: Purification, Blocking Assay, Plasmid Preparation, Recombinant, Lysis, Transfection, SYBR Green Assay, Protease Inhibitor, Methylation, Isolation, Multiplex Assay, Immunodetection, Software, Sequencing

Journal: Immunity

Article Title: m 6 A Modification Prevents Formation of Endogenous Double-Stranded RNAs and Deleterious Innate Immune Responses during Hematopoietic Development

doi: 10.1016/j.immuni.2020.05.003

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Cy3 Streptavidin , VECTOR LABORATORIES , Cat# SA-1300.

Techniques: Purification, Blocking Assay, Plasmid Preparation, Recombinant, Lysis, Transfection, SYBR Green Assay, Protease Inhibitor, Methylation, Isolation, Multiplex Assay, Immunodetection, Software, Sequencing

Journal: Oncotarget

Article Title: Dysfunctional BLK in common variable immunodeficiency perturbs B-cell proliferation and ability to elicit antigen-specific CD4+ T-cell help.

doi: 10.18632/oncotarget.3577

Figure Lengend Snippet: Fig.4: Decreased capacity of L3P-BLK to facilitate B cell receptor-mediated HLA-DR/peptide presentation to CD4+ T -cells. Tetanus toxoid (TT) protein is complexed with anti-IgG F(ab’)2 fragments in ratio 1:2 or 1:4 for 6 hours at 4C (using streptavidin/ biotin conjugation). Paired human B-LCLs expressing common or L3P-BLK variant are loaded (4hrs, 37°C) with 0, 1, or 10 μg anti- IgG-TT complexes. Next, human TT peptide/MHCII-specific CD4+ T-cells are added for co-culture with the B-LCLs (1:1 ratio, O/N, 37°C). B-LCL mediated activation of antigen-specific CD4+ T-cells was measured by analysis of induced CD154 (CD40L) expression. A. CD40L staining in absence of exogenous antigen is set as background levels for both B-LCLs expressing common BLK (upper panel) or L3P-BLK (lower panel). Representative of 3 independent experiments. B. L3P-BLK expressing B-LCL cells (grey) are less able to induce activation of antigen-specific CD4+ T-cells (CD40L high phenotype) compared to common BLK-expressing B-LCL cells (black). Experiments performed on B-LCLs of three independent donors, in at least three independent experiments. Data represented as mean +/- SEM. C. Expression of L3P-BLK does not affect expression of class II MHC molecules (HLA-DR) and co-stimulatory molecules (CD40, CD86, and CD54) in B-LCLs. Data of 3 independent experiments, represented as mean +/- SEM. *P-value <0.05, **P-value <0.01, Two- tailed Wilcoxon-signed rank test.

Article Snippet: Purified Tetanus Toxoid (RIVM, the Netherlands) or DQ-BSA (Life Technologies) was conjugated to Lightening-link streptavidin according to manufactures protocol (Novus Biologicals).

Techniques: Conjugation Assay, Expressing, Variant Assay, Co-Culture Assay, Activation Assay, Staining, Two Tailed Test

Journal: Nucleic Acids Research

Article Title: LC8/DYNLL1 is a 53BP1 effector and regulates checkpoint activation

doi: 10.1093/nar/gkz263

Figure Lengend Snippet: LC8 interacts with 53BP1 on chromatin. (A) Schematic diagram of chromatin interacting protein purification of SFB (S-protein, 2X flag, Streptavidin binding peptide) -tagged-LC8 in HEK293T cells. (B) The top ten list of proteins co-purified with LC8 from mass spectrometry analysis. (C) Protein sequence alignment of LC8 in different species. Protein regions were labeled with secondary structures. Region marked red represents the amino acid residues required for the canonical binding groove formation. (D) Dimerized LC8 structure illustration of the canonical binding groove located between beta 2 and beta 3 sheets. Beta 3 sequence FGSYV is required for directing binding to target protein. Green line represents the putative target binding peptide. (E) Streptavidin pull-down of SFB-LC8 wildtype and SFB-LC8 FGSYV/AAAAA mutant in HEK293T stable cell lines followed by western blotting analysis with Flag and 53BP1 (Novus Biologicals, NB100–304) antibodies.

Article Snippet: Green line represents the putative target binding peptide. ( E ) Streptavidin pull-down of SFB-LC8 wildtype and SFB-LC8 FGSYV/AAAAA mutant in HEK293T stable cell lines followed by western blotting analysis with Flag and 53BP1 (Novus Biologicals, NB100–304) antibodies.

Techniques: Protein Purification, Binding Assay, Purification, Mass Spectrometry, Sequencing, Labeling, Mutagenesis, Stable Transfection, Western Blot

Journal: Nucleic Acids Research

Article Title: LC8/DYNLL1 is a 53BP1 effector and regulates checkpoint activation

doi: 10.1093/nar/gkz263

Figure Lengend Snippet: LC8 promotes 53BP1 irradiation induced foci formation. (A, B) 53BP1 foci is reduced in LC8 KO cells. U2OS cells were irradiated with a Faxitron with 10 Gy and allowed to recover for 1 h. Cells were then fixed, permeabilized and stained with 53BP1 (Novus Biologicals, NB100–304) and γH2AX (Millipore, 05-636) antibodies. Immunofluorescence was analyzed by confocal microscopy. Number of 53BP1 foci per cell were analyzed. (C, D) Cells were treated as in A and B, and stained with 53BP1 (Novus Biologicals, NB100–304) and cyclin A antibodies. Number of 53BP1 foci per cells in both cyclin A-positive group and cyclin A-negative group were analyzed. Yellow dash circle marked cyclin A negative cells. Data represent mean ± SD, ***P< 0.001, ****P< 0.0001. (E, F) U2OS with transient expression of GFP-53BP1 wildtype and GFP-53BP1 GIQ/AAA-TQ/AA were micro-irradiated and quantified. Data represent mean ± S.E.M., *P< 0.05. N = 10 (G) Schematic illustration of 53BP1 mutant fragments used in H and I. (H) Transient co-transfection of SFB-53BP1 ΔOLIGO and myc-53BP1 ΔOLIGO in HeLa wildtype and LC8 KO cells followed by streptavidin pull-down. (I) HEK293T cells were transiently co-transfected with SFB-53BP1 ΔOLIGO and myc-53BP1 ΔOLIGO or SFB-53BP1 ΔOLIGO-GIQ/AAA-TQ/AA and myc-53BP1 ΔOLIGO-GIQ/AAA-TQ/AA. Western blot analysis using antibodies as indicated.

Article Snippet: Green line represents the putative target binding peptide. ( E ) Streptavidin pull-down of SFB-LC8 wildtype and SFB-LC8 FGSYV/AAAAA mutant in HEK293T stable cell lines followed by western blotting analysis with Flag and 53BP1 (Novus Biologicals, NB100–304) antibodies.

Techniques: Irradiation, Staining, Immunofluorescence, Confocal Microscopy, Expressing, Mutagenesis, Cotransfection, Transfection, Western Blot