Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: ?-defensins activate human mast cells via Mas-related Gene-X2 (MrgX2)

doi: 10.4049/jimmunol.1300023

Figure Lengend Snippet: LAD2 mast cells were stably transduced with scrambled shRNA control lentivirus or shRNA lentivirus targeted against MrgX2. (A) Western blotting was performed to determine MrgX2 expression in control and MrgX2 knockdown (KD) cells. (B) shRNA control and MrgX2 KD cells were stimulated with hBD2, hBD3, cortistatin (CST) or C3a and percent degranulation (β-hexosaminidase release) was determined. Data are mean ± SEM of three experiments. Statistical significance was determined by one-way ANOVA with Bonferroni's post test. * indicates p<0.01 and ** indicates p<0.001.

Article Snippet: MrgX2 antibody was purchased from Novus Biologicals (Littleton, CO).

Techniques: Stable Transfection, Transduction, shRNA, Control, Western Blot, Expressing, Knockdown

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: ?-defensins activate human mast cells via Mas-related Gene-X2 (MrgX2)

doi: 10.4049/jimmunol.1300023

Figure Lengend Snippet: (A) RBL-2H3 cells stably expressing MrgX2 were stimulated with buffer, hBD2, hBD3 or cortistatin (CST) for 30 min and β-hexosaminidase release was measured. Data shown are representative of 3 similar experiments. Statistical significance was determined by one-way ANOVA with Bonferroni's post test. * indicates p<0.01 and ** indicates p<0.001. RBL-2H3 cells stably expressing MrgX2 were loaded with Indo-1AM and Ca2+ mobilization in response to (B) hBD2, (C) hBD3 or (D) CST was determined. HEK293 cells stably expressing MrgX2 were loaded with Indo-1AM and Ca2+ mobilization in response to (E) hBD3 or (F) CST was determined. Traces shown are representative of 3 individual experiments.

Article Snippet: MrgX2 antibody was purchased from Novus Biologicals (Littleton, CO).

Techniques: Stable Transfection, Expressing

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: ?-defensins activate human mast cells via Mas-related Gene-X2 (MrgX2)

doi: 10.4049/jimmunol.1300023

Figure Lengend Snippet: (A) BMMCs were transiently transfected with HA tagged MrgX2 (solid line) or control plasmid vector (broken line) and MrgX2 receptor expression level was analyzed using flow cytometry. A representative histogram is shown. (B) Control and MrgX2 expressing BMMCs were incubated with DNP specific mouse IgE (1 μg/mL, 16 h). Cells were exposed to buffer (control), CST, hBD3, mCRAMP or DNP-BSA (10 ng/mL) for 30 minutes and β-hexosaminidase release was measured. LAD2 cells were stimulated with mCRAMP and (C) intracellular Ca2+ mobilization or (D) degranulation was determined. Traces are representative of 3 independent experiments. Bar graphs represent mean ± SEM of three experiments. Statistical significance was determined by one-way ANOVA with Bonferroni's post test. * indicates p<0.01.

Article Snippet: MrgX2 antibody was purchased from Novus Biologicals (Littleton, CO).

Techniques: Transfection, Control, Plasmid Preparation, Expressing, Flow Cytometry, Incubation

Journal: Nature Communications

Article Title: Non-canonical function of DGCR8 in DNA double-strand break repair signaling and tumor radioresistance

doi: 10.1038/s41467-021-24298-z

Figure Lengend Snippet: a Immunoblotting of Drosha, DGCR8, and β-actin in the LM2-DRR (expressing the pLCN DSB Repair Reporter) cell line transduced with DGCR8 shRNA. b Knockdown of DGCR8 decreased HR and NHEJ efficiency in LM2-DRR cells. Two days after co-transfection of I-SceI endonuclease and an exogenous donor for HR (pCAGGS DRR mCherry Donor EF1a BFP) into the DGCR8-knockdown LM2-DRR cells, the percentages of GFP-positive and mCherry-positive cells, gated on BFP-positive cells, were determined by flow cytometry. Repair by HR or NHEJ leads to mCherry or GFP expression. Data were normalized to the control cells. n = 3 biological replicates. c MYC-DGCR8-overexpressing LM2 cells were treated with IR (8 Gy) and cultured for 1 h, followed by pulldown with MYC beads and immunoblotting with the indicated antibodies. d Control and DGCR8-knockdown LM2 cells were treated with IR (8 Gy) and cultured for 1 h, followed by immunoprecipitation with an antibody against RNF168 or RNF8 and immunoblotting with the indicated antibodies. e Chromatin was extracted from LM2 cells that were treated with IR (8 Gy) and cultured for 1 h. The chromatin fractions, with or without MNase treatment, were immunoprecipitated with a DGCR8-specific antibody and immunoblotted with the indicated antibodies. f Quantification of MDC1, RNF8, RNF168, 53BP1, and BRCA1 foci in DGCR8-knockdown LM2 cells. Cells were incubated for 1 h after 2-Gy IR and immunostained with antibodies against γH2AX, MDC1, RNF8, RNF168, 53BP1, and BRCA1 (see representative images in Supplementary Fig. ). n = 3 biological replicates. g Control and DGCR8-knockdown LM2 cells with stable overexpression of FLAG-H2A and RNF8 or RNF168 were transfected with HA-ubiquitin (Ub), treated with IR (8 Gy), and cultured for 8 h, followed by immunoprecipitation with anti-FLAG beads and immunoblotting with antibodies against HA and FLAG. Before immunoprecipitation, lysates were heated at 95 °C for 5 min in the presence of 1% SDS (for denaturing), followed by a 10-fold dilution with lysis buffer and sonication. LE long exposure, SE short exposure. Statistical significance in b and f was determined by a two-tailed unpaired t -test. Error bars are mean ± SEM. n.s . not statistically significant. Source data are provided as a file.

Article Snippet: The cells were washed with phosphate-buffered saline (PBS), fixed with 4% paraformaldehyde, permeabilized with 0.1% Triton X-100 in PBS, blocked with 3% bovine serum albumin in PBS, and incubated with antibodies against γH2AX (1:100, Cell Signaling Technology, #9718S), γH2AX (1:100, BD Biosciences, #560443), DGCR8 (1:100, Abcam, #ab191875), MDC1 (1:200, Bio-Rad, #AHP799), RNF8 (1:200, Proteintech, #14112-1-AP), RNF168 (1:100, Millipore, #ABE367), BRCA1 (1:20, Santa Cruz Biotechnology, #sc-6954), 53BP1 (1:100, Novus Biologicals, #NB100-304), FLAG (1:500, Sigma, #F7425), and MYC (1:500, Santa Cruz Biotechnology, #sc-40, clone 9E10) at 4 °C overnight, followed by incubation with Alexa Fluor 488 goat anti-rabbit IgG (1:1000, Invitrogen, ThermoFisher Scientific, #A-11008), Alexa Fluor 647 donkey anti-sheep IgG (1:1000, Invitrogen, ThermoFisher Scientific, #A-21448), Alexa Fluor 488 goat anti-mouse IgG (1:1000, Invitrogen, ThermoFisher Scientific, #A-11001), Alexa Fluor 594 goat anti-rabbit IgG (1:1000, Invitrogen, ThermoFisher Scientific, #A-11012), and/or Alexa Fluor 594 goat anti-mouse IgG (1:1000, Invitrogen, ThermoFisher Scientific, #A-11005) at room temperature for 1 h. Coverslips were mounted on slides by using anti-fade mounting medium with 4′,6-diamidino-2-phenylindole (DAPI, Vector Laboratories, #H-1200).

Techniques: Western Blot, Expressing, Transduction, shRNA, Knockdown, Cotransfection, Flow Cytometry, Control, Cell Culture, Immunoprecipitation, Incubation, Over Expression, Transfection, Ubiquitin Proteomics, Lysis, Sonication, Two Tailed Test

Journal: Nature Communications

Article Title: Non-canonical function of DGCR8 in DNA double-strand break repair signaling and tumor radioresistance

doi: 10.1038/s41467-021-24298-z

Figure Lengend Snippet: a Immunoblotting of USP36, USP51, and β-actin in parental and radioresistant LM2 cells with and without IR treatment (8 Gy followed by 24-h incubation). b Immunoblotting of DGCR8, USP36, USP51, and β-actin in USP36-knockdown and USP51-knockdown LM2 cells with or without IR treatment (8 Gy followed by 24-h incubation). c Co-IP of endogenous DGCR8 with endogenous USP51. LM2 and LM2-R cells were treated with 8-Gy IR. After 8 h, cells were lysed, immunoprecipitated with a DGCR8-specific antibody, and immunoblotted with antibodies against USP51 and DGCR8. SE short exposure, LE long exposure. d HEK293T cells with stable overexpression of MYC-DGCR8 were co-transfected with SFB-USP51 (wild-type or the C372S mutant) and HA-tagged ubiquitin or the lysine-specific mutant (K48 or K63), and then treated with IR (8 Gy). After 8 h, cells were lysed, denatured, and subjected to immunoprecipitation with anti-MYC beads and immunoblotting with antibodies against HA and MYC. e Knockdown of USP51 decreased HR and NHEJ efficiency in LM2-DRR cells. Two days after co-transfection of I-SceI endonuclease and an exogenous donor for HR (pCAGGS DRR mCherry Donor EF1a BFP) into the USP51-knockdown LM2-DRR cells, the percentages of GFP-positive and mCherry-positive cells, gated on BFP-positive cells, were determined by flow cytometry. Repair by HR or NHEJ leads to mCherry or GFP expression. Data were normalized to the control cells. n = 3 biological replicates. f Quantification of γH2AX, DGCR8, MDC1, RNF8, RNF168, 53BP1, and BRCA1 foci in USP51-knockdown LM2 cells. Cells were incubated for 1 h after 2-Gy IR and immunostained with antibodies against γH2AX, DGCR8, MDC1, RNF8, RNF168, 53BP1, and BRCA1 (see representative images in Supplementary Fig. ). n = 3 biological replicates. g Control and USP51-knockdown LM2 cells with stable overexpression of FLAG-H2A and RNF8 or RNF168 were transfected with HA-ubiquitin (Ub), treated with IR (8 Gy), and cultured for 8 h, followed by immunoprecipitation with anti-FLAG beads and immunoblotting with antibodies against HA and FLAG. Before immunoprecipitation, lysates were heated at 95 °C for 5 min in the presence of 1% SDS (for denaturing), followed by a 10-fold dilution with lysis buffer and sonication. Statistical significance in e and f was determined by a two-tailed unpaired t -test. Error bars are mean ± SEM. Source data are provided as a file.

Article Snippet: The cells were washed with phosphate-buffered saline (PBS), fixed with 4% paraformaldehyde, permeabilized with 0.1% Triton X-100 in PBS, blocked with 3% bovine serum albumin in PBS, and incubated with antibodies against γH2AX (1:100, Cell Signaling Technology, #9718S), γH2AX (1:100, BD Biosciences, #560443), DGCR8 (1:100, Abcam, #ab191875), MDC1 (1:200, Bio-Rad, #AHP799), RNF8 (1:200, Proteintech, #14112-1-AP), RNF168 (1:100, Millipore, #ABE367), BRCA1 (1:20, Santa Cruz Biotechnology, #sc-6954), 53BP1 (1:100, Novus Biologicals, #NB100-304), FLAG (1:500, Sigma, #F7425), and MYC (1:500, Santa Cruz Biotechnology, #sc-40, clone 9E10) at 4 °C overnight, followed by incubation with Alexa Fluor 488 goat anti-rabbit IgG (1:1000, Invitrogen, ThermoFisher Scientific, #A-11008), Alexa Fluor 647 donkey anti-sheep IgG (1:1000, Invitrogen, ThermoFisher Scientific, #A-21448), Alexa Fluor 488 goat anti-mouse IgG (1:1000, Invitrogen, ThermoFisher Scientific, #A-11001), Alexa Fluor 594 goat anti-rabbit IgG (1:1000, Invitrogen, ThermoFisher Scientific, #A-11012), and/or Alexa Fluor 594 goat anti-mouse IgG (1:1000, Invitrogen, ThermoFisher Scientific, #A-11005) at room temperature for 1 h. Coverslips were mounted on slides by using anti-fade mounting medium with 4′,6-diamidino-2-phenylindole (DAPI, Vector Laboratories, #H-1200).

Techniques: Western Blot, Incubation, Knockdown, Co-Immunoprecipitation Assay, Immunoprecipitation, Over Expression, Transfection, Mutagenesis, Ubiquitin Proteomics, Cotransfection, Flow Cytometry, Expressing, Control, Cell Culture, Lysis, Sonication, Two Tailed Test

Journal: Nature Communications

Article Title: Non-canonical function of DGCR8 in DNA double-strand break repair signaling and tumor radioresistance

doi: 10.1038/s41467-021-24298-z

Figure Lengend Snippet: a , b MYC-GFP-, WT DGCR8-, S677A-DGCR8-, and S677D-DGCR8-overexpressing LM2 cells with or without IR treatment ( a , 8 Gy followed by 1-h incubation; b , 8 Gy followed by 8-h incubation) were subjected to pulldown with MYC beads and immunoblotting with the indicated antibodies. c HEK293T cells with stable overexpression of MYC-tagged WT DGCR8, S677A-DGCR8, or S677D-DGCR8 were co-transfected with SFB-USP51 (WT or the C372S mutant) and HA-tagged ubiquitin, and then treated with IR (8 Gy). After 8 h, cells were lysed, denatured, and subjected to immunoprecipitation with anti-MYC beads and immunoblotting with antibodies against HA and MYC. d Quantification of γH2AX, DGCR8, MDC1, RNF8, RNF168, 53BP1, and BRCA1 foci in DRCR8-knockdown LM2 cells with ectopic expression of WT DGCR8, S677A-DGCR8, or S677D-DGCR8. Cells were incubated for 1 h after 2-Gy IR and immunostained with antibodies against γH2AX, DGCR8, MDC1, RNF8, RNF168, 53BP1, and BRCA1 (see representative images in Supplementary Fig. ). n = 3 biological replicates. Statistical significance was determined by a two-tailed unpaired t -test. Error bars are mean ± SEM. e DRCR8-knockdown LM2 cells with ectopic expression of WT DGCR8 or the S677A mutant were transduced with FLAG-H2A and RNF8 or RNF168. The cells were then transfected with HA-ubiquitin (Ub), treated with IR (8 Gy), and cultured for 8 h, followed by immunoprecipitation with anti-FLAG beads and immunoblotting with antibodies against HA and FLAG. Before immunoprecipitation, lysates were heated at 95 °C for 5 min in the presence of 1% SDS (for denaturing), followed by a 10-fold dilution with lysis buffer and sonication. LE long exposure, SE short exposure. Source data are provided as a file.

Article Snippet: The cells were washed with phosphate-buffered saline (PBS), fixed with 4% paraformaldehyde, permeabilized with 0.1% Triton X-100 in PBS, blocked with 3% bovine serum albumin in PBS, and incubated with antibodies against γH2AX (1:100, Cell Signaling Technology, #9718S), γH2AX (1:100, BD Biosciences, #560443), DGCR8 (1:100, Abcam, #ab191875), MDC1 (1:200, Bio-Rad, #AHP799), RNF8 (1:200, Proteintech, #14112-1-AP), RNF168 (1:100, Millipore, #ABE367), BRCA1 (1:20, Santa Cruz Biotechnology, #sc-6954), 53BP1 (1:100, Novus Biologicals, #NB100-304), FLAG (1:500, Sigma, #F7425), and MYC (1:500, Santa Cruz Biotechnology, #sc-40, clone 9E10) at 4 °C overnight, followed by incubation with Alexa Fluor 488 goat anti-rabbit IgG (1:1000, Invitrogen, ThermoFisher Scientific, #A-11008), Alexa Fluor 647 donkey anti-sheep IgG (1:1000, Invitrogen, ThermoFisher Scientific, #A-21448), Alexa Fluor 488 goat anti-mouse IgG (1:1000, Invitrogen, ThermoFisher Scientific, #A-11001), Alexa Fluor 594 goat anti-rabbit IgG (1:1000, Invitrogen, ThermoFisher Scientific, #A-11012), and/or Alexa Fluor 594 goat anti-mouse IgG (1:1000, Invitrogen, ThermoFisher Scientific, #A-11005) at room temperature for 1 h. Coverslips were mounted on slides by using anti-fade mounting medium with 4′,6-diamidino-2-phenylindole (DAPI, Vector Laboratories, #H-1200).

Techniques: Incubation, Western Blot, Over Expression, Transfection, Mutagenesis, Ubiquitin Proteomics, Immunoprecipitation, Knockdown, Expressing, Two Tailed Test, Transduction, Cell Culture, Lysis, Sonication

Journal: Neuro-Oncology

Article Title: miR-423-5p contributes to a malignant phenotype and temozolomide chemoresistance in glioblastomas

doi: 10.1093/neuonc/now129

Figure Lengend Snippet: miR-423-5p overexpression promotes cell proliferation, angiogenesis, and invasion. (A) The overexpression of miR-423-5p promoted cell proliferation, which was rescued upon induction of the expression of exogenous ING-4 in both U87 and U251 cells. *indicates significant difference compared with control, #indicates significant difference compared with miR-423-5p/ING-4 treatment at P < .01. (B) Cell-cycle assay results of U87 and U251 glioma cells 3 days after transfection with miR-423-5p or control miR (monitored by flow cytometry). (C) Western blot analysis was performed to indicate the regulation of cell-cycle–regulated proteins, cyclin D1, cyclin E1, and p-RB by miR-423-5p in U87 cells. (D) Apoptosis assay results of U87 and U251 glioma cells 3 days after transfection with the miR-423-5p inhibitor or the control miR were monitored by flow cytometry. (E) Tube formation of human brain microvessel endothelial cells (HBMVECs) transfected with miR-423-5p or control miR was monitored by Matrigel-coated plates with conditioned medium from the indicated cells. Scale bar = 300 μm. (F) miR-423-5p overexpression promoted cell invasion in U87 cells. Cells were transfected with miR-423-5p, which was followed by ING-4 transfection. Scale bar = 100 μm. *P < .05 and ***P < .001.

Article Snippet: The antibodies that were used included ING-4, MMP9, p53, BCL-2, BAX (Abcam), CD133 (Amersham), Nestin (BD), cyclin D1, cyclin E1, p-RB Ser780, GFAP, BMI1, MMP2, EGFR, PDGFR, Akt and pAkt Ser473 (Cell Signaling), ERK and Phospho-ERK1 (T202/Y204)/ERK2 (T185/Y187), Sox2 (R&D Systems), and GAPDH (Sigma Aldrich).

Techniques: Over Expression, Expressing, Control, Cell Cycle Assay, Transfection, Flow Cytometry, Western Blot, Apoptosis Assay

Journal: Frontiers in Immunology

Article Title: Orthotopic and Heterotopic Murine Models of Pancreatic Cancer Exhibit Different Immunological Microenvironments and Different Responses to Immunotherapy

doi: 10.3389/fimmu.2022.863346

Figure Lengend Snippet: NKG2D CAR-T rescue impaired CAR-T cell activity via eliminating MDSCs. (A) Schematic diagram of NKG2D-CAR (Upper) and flow cytometry assay of transfection efficiency (Down). (B) Cytotoxicity assay of NKG2D CAR-T to Rae1 positive CHO cell at indicated E:T ratio. E:T, effector-to-target ratio. (n=6 compared with Control CAR-T group, ***P<0.001). (C) Cytotoxicity assay of NKG2D CAR-T to murine MDSCs at E:T=1:1. MDSCs were pre-labeled by CSFE. CSFE + 7AAD + cells indicate specific killed MDSCs. (D) Survival of orthotopic tumor recipients after NKG2D CAR-T transfer. The green arrows indicate NKG2D CAR-T cells administration. (E) Representative IHC images of Gr1 staining in orthotopic tumor 2 days after NKG2D CAR-T transfer. Scale bar=50μm. (F) Schematic outline of experimental approaches and analyses. (G) Survival of orthotopic tumor recipients after combination of NKG2D CAR-T and αMUC1 CAR-T. The green arrows indicate NKG2D CAR-T cells administration; the orange arrows indicate αMUC1 CAR-T cells administration. (***P<0.001). (H) Schematic diagram of bicistronic CAR (Upper) and flow cytometry assay of transfection efficiency (Down). (I) Survival of orthotopic tumor recipients after bicistronic CAR-T transfer. The orange arrow indicates CAR-T cells administration (***P<0.001).

Article Snippet: The extracellular (EC) and transmembrane (TM) regions of mice NKG2D (NM_001083322.2) were cloned form commercial plasmid (SinoBiological, MG57340). scFv sequence of CD19 was from patent US20210395364A1 in VL-(G 4 S) 3 -VH form. scFv sequence of MUC1 was from patent US10239950B2 in VL-(G 4 S) 3 -VH form.

Techniques: Activity Assay, Flow Cytometry, Transfection, Cytotoxicity Assay, Labeling, Staining

Journal: bioRxiv

Article Title: Prognostically favorable immune responses to ovarian cancer are distinguished by self-reactive intra-epithelial plasma cells

doi: 10.1101/2025.05.12.652297

Figure Lengend Snippet: (A) Silver-stained (SS) gel of immunoprecipitation (IP) output samples showing unique bands immunoprecipitated by TIL-B rAb D-1-m1 (red boxes) relative to the control (black box). Labels indicate the IP inputs (No Ab versus TIL-B rAb D-1-m1 and buffer versus OVCAR3 lysate) related to each IP output sample analysed on the gel. Gel pieces corresponding to the red/black boxes were excised and analyzed by mass spectrometry (MS). (B) Top 10 proteins (ranked by peptide counts) uniquely identified by MS in both replicates of the TIL-B rAb D-1-m1 + OVCAR3 IP output samples relative to controls. (C) ELISA with TIL-B rAb D-1-m1 and recombinant KDM4a, GST, and BSA proteins. (D) IP-Western blot (WB) with IP output samples from TIL-B rAb D-1-m1 (or no Ab) + OVCAR3 lysate (or buffer) probed with a commercial anti-KDM4a antibody. (E) IP-SS gel of 10% of the total IP output samples with TIL-B rAb B-13-m1 (or no Ab or isotype control rAb) and OVCAR3 lysate (or buffer) input. (F) Top 10 proteins (ranked by peptide counts) uniquely identified by MS in the TIL-B rAb B-13-m1 + OVCAR3 IP output sample relative to controls. (G) ELISA with TIL-B rAb B-13-m1 and recombinant NuMA, GST, and BSA proteins. (H) IP-WB with TIL-B rAb B-13-m1 (or no rAb) + OVCAR3 (or buffer) IP output samples probed with a commercial anti-NuMA antibody. (I) Discovery of DYNLT2B as the target antigen of TIL-B rAb B-9-m1 by phage-based cDNA immunoscreening (modified SEREX). (J) ELISA with TIL-B rAb B-9-m1 and recombinant DYNLT2B, GST, and BSA proteins. (K) Examples of immunohistochemistry (IHC) staining on patient-derived tissues with commercial antibodies against all three identified TIL-B target antigens. BSA: Bovine serum albumin. GST: Glutathione S transferase.

Article Snippet: Antigens were sourced commercially as follows: KDM4a (Signal Chem, K424-31G-50), NuMA (LSBio, LS-G24789-50), bovine serum albumin (BSA) (Thermo Fisher, 23200), glutathione-S-transferase (GST) (Signal Chem, G52-30U).

Techniques: Staining, Immunoprecipitation, Control, Mass Spectrometry, Enzyme-linked Immunosorbent Assay, Recombinant, Western Blot, Modification, Immunohistochemistry, Derivative Assay

Journal: bioRxiv

Article Title: Prognostically favorable immune responses to ovarian cancer are distinguished by self-reactive intra-epithelial plasma cells

doi: 10.1101/2025.05.12.652297

Figure Lengend Snippet: (A) Lineages of selected TIL-B clones. Mutant (m) number identifies individual clonotypes. Number of circles = number of cells. Numbers on branches indicate the total number of nucleotide mutations between relevant clonotypes. Bolded clonotypes are the dominant clonotype used in initial analyses. (B) Heatmap summarizing TIL-B rAb reactivity for all 35 clonally related rAbs as measured by intracellular and cell surface flow cytometry. Red arrows in panels A and B indicate the individual clonotype within each lineage that demonstrated the highest reactivity (measured by total median MFI) across all cell types evaluated. (C-E) ELISA data with rAbs from TIL-B clonal lineages D-1 (C) , B-13 (D) , and B-9 (E) and recombinant KDM4a, NuMA, DYNLT2B, GST, and BSA proteins. BSA: Bovine serum albumin. GST: Glutathione S transferase. MFI: Median fluorescence intensity.

Article Snippet: Antigens were sourced commercially as follows: KDM4a (Signal Chem, K424-31G-50), NuMA (LSBio, LS-G24789-50), bovine serum albumin (BSA) (Thermo Fisher, 23200), glutathione-S-transferase (GST) (Signal Chem, G52-30U).

Techniques: Clone Assay, Mutagenesis, Flow Cytometry, Enzyme-linked Immunosorbent Assay, Recombinant, Fluorescence

Journal: bioRxiv

Article Title: Prognostically favorable immune responses to ovarian cancer are distinguished by self-reactive intra-epithelial plasma cells

doi: 10.1101/2025.05.12.652297

Figure Lengend Snippet: (A) Heatmap summarizing polyreactivity (polyrx) ELISA data for all 51 TIL-B rAbs. (B) ELISA with serum from patient (PT) D and 15 healthy donors (HD) against recombinant KDM4a, BSA, and GST. **** = p < 0.0001 as measured by two-way ANOVA. (C) ELISA with serum from PT B and 15 HD and recombinant NuMA, BSA, and GST. (D) Anti-nuclear antibody (ANA) titres from immunofluorescence staining with HGSC patient serum (N=10) in reference to previously published ANA results for HD and autoimmune rheumatic disease (ARD) patients . (E) Bar plots showing the distribution of TIL-Bs across B cell (BC) and plasma cell (PC) phenotypic clusters within different B cell receptor (BCR) groups based on expansion status or related rAb reactivity. P values measured by Fisher’s exact test. BSA: Bovine serum albumin. dsDNA: Double-stranded DNA. Exp: Expanded. GST: Glutathione S transferase. Lin: Lineage. LPS: Lipopolysaccharide. ssDNA: single-stranded DNA. Tx: Treatment.

Article Snippet: Antigens were sourced commercially as follows: KDM4a (Signal Chem, K424-31G-50), NuMA (LSBio, LS-G24789-50), bovine serum albumin (BSA) (Thermo Fisher, 23200), glutathione-S-transferase (GST) (Signal Chem, G52-30U).

Techniques: Enzyme-linked Immunosorbent Assay, Recombinant, Immunofluorescence, Staining, Clinical Proteomics

Journal: Cellular and Molecular Life Sciences

Article Title: Tumor cell-intrinsic and tumor microenvironmental conditions co-determine signaling by the glycoimmune checkpoint receptor Siglec-7

doi: 10.1007/s00018-023-04816-6

Figure Lengend Snippet: CD43-expressing cells can induce chSiglec-7 signaling under the condition of sufficient sialic acid availability. a HEK293-6E cells were transfected to express CD43 or were mock-transfected and CD43 expression was determined by flow cytometry and compared to the respective isotype control. b Siglec-7 ligand expression was determined by a recombinant human Siglec-7 Fc staining, measured by flow cytometry. c chSiglec-7 or d chSiglec-7R124A expressing Jurkat/MA cells were co-cultured (1:1) with the CD43 or mock transfected HEK293-6E cells and luminescence was assessed using a luciferase assay (n = 5). c , d Nested data are presented of 4 experiments, each performed in duplicate. e Siglec-7 Fc binding to parental IMR-32 cells or CD43 + IMR-32 cells was assessed by flow cytometry and the MFI was quantified. f chSiglec-7 or g chSiglec-7R124A expressing Jurkat/MA cells were co-cultured (1:1) with parental IMR-32 cells or CD43 + IMR-32 cells and luminescence as assessed using a luciferase assay (n = 2). IMR-32 cells were pretreated with Ac 5 Neu5Ac or DMSO as vehicle control for 3 days, which was refreshed on day 2. Representative data are shown of two independent experiments and data present mean ± SD

Article Snippet: The open reading frame of human CD43 was cloned from a pGEM-T vector with the human CD43 open reading frame (SinoBiological, HG13108-G) into the pEGFP-N3 vector, replacing EGFP.

Techniques: Expressing, Transfection, Flow Cytometry, Recombinant, Staining, Cell Culture, Luciferase, Binding Assay

Journal: Frontiers in Immunology

Article Title: The Sez6 Family Inhibits Complement by Facilitating Factor I Cleavage of C3b and Accelerating the Decay of C3 Convertases

doi: 10.3389/fimmu.2021.607641

Figure Lengend Snippet: Full Length Sez6L2, Sez6, and Sez6L inhibit C3b/iC3b opsonization of CHO cells by the classical pathway. (A, B) Sez6L2 inhibits C3b/iC3b opsonization at a range of serum concentrations. CHO cells were transfected with plasmids for GFP alone or with Myc-tagged Sez6L2 (M-Sez6L2) or His-tagged DAF (H-DAF). CHO cells were coated with antibodies and exposed to 0-20% C5-depleted human serum for one hour and then immuno-stained with anti-C3b/iC3b antibodies and analyzed by flow cytometry. One experiment is shown that is representative of two independent experiments. (B) C3b/iC3b on GFP transfected cells with or without M-Sez6L2 or H-DAF at 15% serum. ANOVA (P=0.0016; F(2,6)=22.51). N=3; one experiment with three replicates (representative of 3+ independent experiments). (C) Schematic of Sez6L2, Sez6, and Sez6L protein domain structures. (D–I) CHO cells were transfected with the indicated Myc-tagged cDNAs and processed as outlined in A with 15% C5 depleted serum, except that an anti-Myc antibody was used in place of GFP to identify transfected and expressing CHO cells. (D) 5% Contour plots of C3b/iC3b versus Myc fluorescence (top layer) and C3b/iC3b fluorescence histograms (bottom layer) of the same samples normalized to mode and compared to baseline cells not exposed to serum. For Contour plots, boxed regions highlight cells designated as Myc-positive (top box) and Myc-negative (lower box) populations. For C3b/iC3b histograms, dark grey, solid line population = Myc-positive cells; Light grey, dotted line population= Myc-negative cells; White, dashed grey line population = baseline. Representative of 4+ independent experiments. (E) Quantification of the average median C3b/iC3b fluorescence intensity from Myc-positive and Myc-negative cells within each sample. Statistics = t-tests. N=3 (one experiment with three replicates; Representative of 4+ independent experiments). (F) Average median C3b/iC3b fluorescence intensities after normalization to the Myc-negative cells from each experimental group. ANOVA between Myc-positive cell populations (p<0.001; F(4, 15)=64.53). Sez6L2 inhibits C3b/iC3b opsonization at a level comparable to positive control MCP. Sez6 is a stronger complement inhibitor than Sez6L2 and Sez6L is a weaker inhibitor. (F) Average median Myc fluorescence intensity from Myc-positive cells. ANOVA (p<0.001; F(4, 15)=36.79). (G) Average % of Myc-positive cells in each experimental group (ANOVA, p=0.115; F(4, 15)=2.224). For sections (F–H) , N=4 (four independent experiments). (I) Sez6 blocks complement opsonization more efficiently than Sez6L2 and Sez6L even when comparing similar levels of Myc surface expression. Average C3b/iC3b median fluorescence intensity normalized to internal Myc-negative populations for M-Sez6, M-Sez6L2, and M-Sez6L samples shown relative to the Myc median fluorescence intensity. N=3 (one experiment with three replicates, Representative of three independent experiments). For all graphs *p < 0.05; **p < 0.01; # p < 0.001 for all Myc-positive groups compared to M-CR2.

Article Snippet: Alternatively, cells were transfected with a GFP expression plasmid or co-transfected with GFP and M-SEZ6L2 or HIS-tagged DAF (H-DAF; Sino Biological HG10101-NH; modified from NCBI RefSeq NM_000574.3).

Techniques: Transfection, Staining, Flow Cytometry, Expressing, Fluorescence, Positive Control