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

Journal: Cellular and Molecular Immunology

Article Title: γδ T-cell autoresponses to ectopic membrane proteins: a new type of pattern recognition

doi: 10.1038/s41423-025-01258-x

Figure Lengend Snippet: Two CDR3δ peptides, GTM and OT3, with tumor binding specificity served as probes to screen the protein ligands of human γδ TCRs. a and b. Immunohistochemical staining of a tumor tissue array with the biotin-labeled GTM peptide a and biotin-labeled OT3 peptide b followed by HRP-conjugated streptavidin. The binding was visualized using DAB as the substrate (brown) (400×). Cancer: tumor tissue; paracancerous: paracarcinoma tissues. Scale bar, 50 μm. GTM and OT3 can specifically bind to several tumor cell lines. Fifty-three types of human tumor cell lines were stained with biotinylated (bio)-GTM c or (bio)-OT3 peptide d and APC-conjugated streptavidin. The binding activity was measured by flow cytometry. The flow data were standardized by the ratio between the mean fluorescence intensity of the peptide conjugate and the secondary antibody alone. The binding strength of the synthetic peptide was compared with that of normal human peripheral blood mononuclear cells (PBMCs). MST analysis of the binding activities of the GTM peptide e and OT3 peptide f to known γδ TCR-recognized stress-inducible ligands. The Kd value for each protein was calculated. g Schematic diagram of OT3- and GTM-γδ CAR molecules. A single-chain Vγ9-Vδ2 or Vγ4-Vδ1 structure, which is composed of a Vδ2 TCR with the tumor-specific CDR3δ sequence OT3 or Vδ1 TCR with the tumor-specific CDR3δ sequence GTM, forms the extracellular region of the γδ CAR. The transmembrane and intracellular segments of a classical third-generation CAR (CD8-CD28-4-1BB-CD3ζ) were linked to the Vγ-Vδ sequence. γδ CARs were cloned and inserted into the lentiviral vector cFUGW. Flow cytometry was used to assess the expression of the activation marker CD69, the secretion of TNF-α h and the cytotoxicity-related molecules granzyme B and perforin i in γδ CAR-T cells after coculture with OVCAR-8 cells in vitro. The statistical data are representative of three independent experiments and are expressed as the means ± SDs. *, p < 0.05; **, p < 0. 01. Cytotoxicity of OT3 CAR-T cells j and GTM CAR-T cells k in different tumor cells in vitro. Cytotoxicity was analyzed with an LDH cytotoxicity detection kit. The data are representative of three independent experiments and are expressed as the means ± SDs. OT3 CAR-αβ T cells exhibit strong cytotoxic activity against tumors in an NSG mouse model. OVCAR-8-transplanted NSG mice were treated with GFP-αβ T cells or OT3 CAR-αβ T cells. Tumor growth in every mouse at each time point was measured by assessing the radiance of the tumor cells l . The tumor growth curve m and survival curve n of each group are shown

Article Snippet: In the OT3 protein microarray, OT3 peptide or biotin, as the primary antibody, was diluted, added, and incubated at 4 °C for 12 h. Cy3-conjugated streptavidin (Bioss) was used to probe the primary antibodies bound to the slide at RT for 1 h. For all the assays, a rabbit anti-GST monoclonal antibody (CST) was added and incubated at RT for 1 h. An Alexa Fluor 647-conjugated goat anti-rabbit IgG (CST) secondary antibody was added and incubated at RT for 1 h. Finally, the microarray slides were scanned with a GenePix 4000B fluorescent microarray scanner (GBI).

Techniques: Binding Assay, Immunohistochemical staining, Staining, Labeling, Activity Assay, Flow Cytometry, Fluorescence, Sequencing, Clone Assay, Plasmid Preparation, Expressing, Activation Assay, Marker, In Vitro

Journal: bioRxiv

Article Title: High-speed Intracellular Temperature Mapping Reveals the Existence of Non-Conductive Dissipation of Energy by Heating

doi: 10.1101/2024.08.29.610413

Figure Lengend Snippet: The temperature-dependent fluorescence intensity decrease (left). An exponential approximation (Fluorescence intensity = 2.076e -0.0306 T , where T indicates the temperature; R = 0.99) was used for the estimation of temperature change (Δ T ). Comparison of normalized temperature relaxation after continuous heating with IR laser irradiation (5 s) between the result by FPT (black, ) and that by Cy3 (IR laser: 0.55 mW, red) (right). Scale bars represent indicated temperature changes. Data are presented as mean ± SE [ n = 35 (Cy3), 29 (FPT) cells]. Inset in the right panel indicates area under the curve (AUC) of normalized temperature relaxation. NS indicates not significant.

Article Snippet: Microinjection of FPT , Rhodamine B-labeled dextran (molecular weight 10,000; Thermo Fisher Scientific), and Cy3 tagged Streptavidin (FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan) into living cells was performed with Femtojet (Eppendorf, Hamburg, Germany), controlled by a micromanipulator (Eppendorf).

Techniques: Fluorescence, Comparison, Irradiation