Journal: Immunity

Article Title: Excessive Polyamine Generation in Keratinocytes Promotes Self-RNA Sensing by Dendritic Cells in Psoriasis.

doi: 10.1016/j.immuni.2020.06.004

Figure Lengend Snippet: Figure 5. Polyamines Enable Self-RNA Sensing by Myeloid DCs (A) Statistics of the flow cytometric analysis of DCs cultured overnight in dead keratinocyte supernatants generated from Pp6fl/fland K5. Pp6fl/flmice (n = 3). (B) Study protocol to identify proteins or peptides in a complex with spermidine or spermine in human blood plasma samples. (C) LC-MS/MS-based detection of the abundance of HNRNPA1226-237 in the immunoprecipitates pulled by anti-spermidine from the blood plasma of patients with psoriasis (n = 3, Pso) and healthy donors (n = 3, Nor). Immunoprecipitates pulled by rabbit immunoglobulin G (IgG) from the blood plasma of patients with psoriasis (n = 3, IgG) served as negative controls. UD, undetected. (D) The human HNRNPA1 protein functional domains described in UniProt. (E) Confocal microscopic images of mouse BMDCs treated with 488-labeled RNA, TMR-labeled Hnrnpa1226-237 and 2-(methylamino) benzoic-acid-labeled spermidine, with the cells co-stained with MHC class II. (F) Agarose gel showing the degradation of RNA treated with the indicated reagents. (G) ELISA quantification of IL-6 in mouse BMDC cultures stimulated with the indicated reagents (n = 3). (H) ELISA quantification of IL-6 in human moDC cultures stimulated with the indicated reagents (n = 5). Data in (A and E–H) are representative of three independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001, two-tailed Student’s t test (mean ± SEM). See also Figures S5 and S6.

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Chemicals, Peptides, and Recombinant Proteins Recombinant Mouse GM-CSF R&D Systems Cat#415-ML Recombinant Mouse IL-4 R&D Systems Cat#404-ML Recombinant Mouse IL-23 R&D Systems Cat#1887-ML-010 Recombinant Human GM-CSF R&D Systems Cat#215-GM Recombinant Human IL-4 R&D Systems Cat#204-IL Recombinant Murine Flt3-Ligand PeproTech Cat#250-31L L-arginine Sangon Biotech Cat#A600205 L-ornithine Sigma-Aldrich Cat#O6503 L-proline Sangon Biotech Cat#A600923 Creatine Sangon Biotech Cat#A600326 L-citrulline Sangon Biotech Cat#A604057 Spermidine Sigma-Aldrich Cat#S0266 nor-NOHA Cayman Cat#1140844-63-8 Oligomycin Abcam Cat#141829 DFMO Sigma-Aldrich Cat#D193 IMQ cream MedShine Cat#120503 Halometasone cream Bright Future Pharmaceutical Laboratories, Ltd. N/A Ribonuclease A Sigma-Aldrich Cat#R6513 HNRNPA1226-237 Genescript N/A TMR-labeled HNRNPA1226-237 Genescript N/A Krt1017-28 Genescript N/A CellTrace Violet Invitrogen Cat#C34557 ionomycin Sigma-Aldrich Cat#I9657 PMA Sigma-Aldrich Cat#79346 GolgiPlug BD Biosciences Cat#555029 Critical Commercial Assays Peroxidase Substrate Kit ZSGB-BIO Cat#ZLI-9018 Protein A/G magnetic beads Merck Millipore Cat#LSKMAGAG02 CD14 Microbeads Miltenyi Biotec Cat#130-050-201 TruSeq RNA sample preparation kit Illumina Cat#FC-122-1001 TruSeq paired-end cluster kit v3-cBot-HS Illumina Cat#PE-401-3001 M-MLV First-Strand Synthesis Kit Invitrogen Cat#C28025-021 Hieff qPCR SYBR Green Master Mix Yeasen Biotech Cat#11202ES03 Minute Endosome Isolation and Cell Fractionation Kit Invent Biotech Cat#ED-028 Mouse Cytokine Array GS4000 RayBiotech Cat#GSM-CAA-4000-1 Mouse IgE ELISA MAX Deluxe BioLegend Cat#432404 Mouse IL-6 ELISA kit NeoBioscience Cat#EMC004.96 Mouse TNF-a ELISA kit NeoBioscience Cat#EMC102a.96 Mouse IFN-alpha ELISA Kit R&D Systems Cat#42120-1 Human IL-6 ELISA Kit MultiSciences Cat#70-EK106/2-96 Human TNF-a High Sensitivity ELISA Kit MultiSciences Cat#70-EK182HS-96 SimpleCHIP enzymatic chromatin immunoprecipitation kit Cell Signaling Technology Cat#9002 Duolink In Situ Red Starter Kit Mouse/Rabbit Sigma-Aldrich Cat#DUO92101-1KT OxiSelect?

Techniques: Cell Culture, Generated, Clinical Proteomics, Liquid Chromatography with Mass Spectroscopy, Functional Assay, Labeling, Staining, Agarose Gel Electrophoresis, Enzyme-linked Immunosorbent Assay, Two Tailed Test

Journal: Immunity

Article Title: Excessive Polyamine Generation in Keratinocytes Promotes Self-RNA Sensing by Dendritic Cells in Psoriasis.

doi: 10.1016/j.immuni.2020.06.004

Figure Lengend Snippet: Figure 6. Polyamine-Mediated Self-RNA Sensing Requires Endosomal Tlr7 (A) ELISA quantification of IL-6 in WT and Tlr7-deficient BMDC cultures stimulated with the indicated reagents (n = 3). (B) qPCR detection of human 28S rRNA in endosomes isolated from mouse BMDCs treated with the indicated reagents (n = 7). The fold changes relative to the self-RNA group are presented. (C) Immunoblotting analysis of IkappaB-z expression in nuclear fractions of BMDCs treated with the indicated reagents for 4 h. (D and E) RNAscope of Krt14 combined with immunofluorescent labeling of Tlr7 and spermidine on DCs isolated from mouse skin treated with IMQ or untreated (UT). Scale bars, 100 mm (D) or 3 mm (E). (F) GSEA of genes in CLEC9A+WDFY4+ cDC1 and in CD1C+SIRPA+ cDC2 or moDC from human psoriatic skin relative to healthy skin with the genes induced in R848 stimulated DCs enriched (GSE2706). GSEA of skin DCs in lesional K5. Pp6fl/flskin relative to normal mouse skin with the genes induced in R848 stimulated DCs enriched (GSE2706). Data in (A–E) are representative of two independent experiments. *p < 0.05, **p < 0.01, two-tailed Student’s t test (mean ± SEM). See also Figure S7.

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Chemicals, Peptides, and Recombinant Proteins Recombinant Mouse GM-CSF R&D Systems Cat#415-ML Recombinant Mouse IL-4 R&D Systems Cat#404-ML Recombinant Mouse IL-23 R&D Systems Cat#1887-ML-010 Recombinant Human GM-CSF R&D Systems Cat#215-GM Recombinant Human IL-4 R&D Systems Cat#204-IL Recombinant Murine Flt3-Ligand PeproTech Cat#250-31L L-arginine Sangon Biotech Cat#A600205 L-ornithine Sigma-Aldrich Cat#O6503 L-proline Sangon Biotech Cat#A600923 Creatine Sangon Biotech Cat#A600326 L-citrulline Sangon Biotech Cat#A604057 Spermidine Sigma-Aldrich Cat#S0266 nor-NOHA Cayman Cat#1140844-63-8 Oligomycin Abcam Cat#141829 DFMO Sigma-Aldrich Cat#D193 IMQ cream MedShine Cat#120503 Halometasone cream Bright Future Pharmaceutical Laboratories, Ltd. N/A Ribonuclease A Sigma-Aldrich Cat#R6513 HNRNPA1226-237 Genescript N/A TMR-labeled HNRNPA1226-237 Genescript N/A Krt1017-28 Genescript N/A CellTrace Violet Invitrogen Cat#C34557 ionomycin Sigma-Aldrich Cat#I9657 PMA Sigma-Aldrich Cat#79346 GolgiPlug BD Biosciences Cat#555029 Critical Commercial Assays Peroxidase Substrate Kit ZSGB-BIO Cat#ZLI-9018 Protein A/G magnetic beads Merck Millipore Cat#LSKMAGAG02 CD14 Microbeads Miltenyi Biotec Cat#130-050-201 TruSeq RNA sample preparation kit Illumina Cat#FC-122-1001 TruSeq paired-end cluster kit v3-cBot-HS Illumina Cat#PE-401-3001 M-MLV First-Strand Synthesis Kit Invitrogen Cat#C28025-021 Hieff qPCR SYBR Green Master Mix Yeasen Biotech Cat#11202ES03 Minute Endosome Isolation and Cell Fractionation Kit Invent Biotech Cat#ED-028 Mouse Cytokine Array GS4000 RayBiotech Cat#GSM-CAA-4000-1 Mouse IgE ELISA MAX Deluxe BioLegend Cat#432404 Mouse IL-6 ELISA kit NeoBioscience Cat#EMC004.96 Mouse TNF-a ELISA kit NeoBioscience Cat#EMC102a.96 Mouse IFN-alpha ELISA Kit R&D Systems Cat#42120-1 Human IL-6 ELISA Kit MultiSciences Cat#70-EK106/2-96 Human TNF-a High Sensitivity ELISA Kit MultiSciences Cat#70-EK182HS-96 SimpleCHIP enzymatic chromatin immunoprecipitation kit Cell Signaling Technology Cat#9002 Duolink In Situ Red Starter Kit Mouse/Rabbit Sigma-Aldrich Cat#DUO92101-1KT OxiSelect?

Techniques: Enzyme-linked Immunosorbent Assay, Isolation, Western Blot, Expressing, RNAscope, Labeling, Two Tailed Test

Journal: Cell Reports Medicine

Article Title: Immunization with a self-assembling nanoparticle vaccine displaying EBV gH/gL protects humanized mice against lethal viral challenge

doi: 10.1016/j.xcrm.2022.100658

Figure Lengend Snippet:

Article Snippet: Goat anti-human IgG , SouthernBiotech , Cat# 1030–01; RRID: AB_2794290.

Techniques: Recombinant, Virus, Cloning, Saline, Plasmid Preparation, Emulsion, Adjuvant, Binding Assay, Transfection, Magnetic Beads, Software, Real-time Polymerase Chain Reaction, Cytometry, Luminex, High Performance Liquid Chromatography, Multi-Angle Light Scattering, Refractive Index

Journal: Biology Methods & Protocols

Article Title: Protocol for isolation and expansion of natural killer cells from human peripheral blood scalable for clinical applications

doi: 10.1093/biomethods/bpaf015

Figure Lengend Snippet: List of required materials for natural killer cell isolation, expansion, and cryopreservation.

Article Snippet: MACS multi-stand , Miltenyi Biotec , 130-042-303.

Techniques: Cell Isolation, Irradiation, Transferring

Journal: Frontiers in Immunology

Article Title: Baseline AHR expression shapes immune response to pharmacological modulation in PBMCs from pancreatic cancer patients

doi: 10.3389/fimmu.2025.1655258

Figure Lengend Snippet: AHR pathway modulation alters IL1B, IL4, IL6, and IL10 expression (FC, Luminex) in PDAC PBMCs. Cytokine expression was assessed at mRNA, soluble, and intracellular levels in M1 and M2 monocytes following treatment with Carbidopa, Tapinarof, or BAY. Samples were stratified by baseline AHR expression. CTRL refers to untreated PDAC PBMCs stimulated with LPS but without AHR modulators or tumor co-culture. Healthy indicates LPS-stimulated PBMCs from non-cancer donors, and co-cultured refers to healthy PBMCs indirectly co-cultured with BxPC-3 cells. Sample numbers (n) varied between assays and AHR expression groups due to differences in PBMC yield and viability (typically n = 1–13 for PDAC High/Medium AHR, n = 2–13 for PDAC Low AHR, and n = 11–14 for healthy donors; see for details). Color coding: red = upregulated; blue = downregulated; white = no significant change; ↑/↓ indicate directional changes; Δ – significant vs. CTRL PDAC; *p < 0.05 vs. healthy; **p < 0.05 vs. co-cultured; NS, not significant.

Article Snippet: The concentrations of cytokines IL-1β, IL4, and IL10 were quantified in the culture media collected after 24 h of healthy and PDAC PBMCs incubation using magnetic bead–based multiplex assays (Human Cytokine Premixed Multi-Analyte Kit, R&D Systems, Minneapolis, MN, USA) and analyzed using a Luminex 100 analyzer (Luminex Corporation, Cat# L100-XPONENT, Austin, TX, USA; RRID: SCR_018025).

Techniques: Expressing, Luminex, Co-Culture Assay, Cell Culture

Journal: Cell

Article Title: LRRC37B is a human modifier of voltage-gated sodium channels and axon excitability in cortical neurons

doi: 10.1016/j.cell.2023.11.028

Figure Lengend Snippet: LRRC37B expression in the mammalian cerebral cortex, related to Figure 1 (A) LRRC37 transcripts detected by bulk RNA-seq in the cerebral cortex in mouse, macaque, and human (expression in RPKM, reads per kilobase million; median + SE median; from Cardoso-Moreira et al. ). (B) Expression of LRRC37B (complementary to Figures 1 D and 1E) in chimpanzee and human cells of the dorsolateral prefrontal cortex (expression in logCPM, log counts per million, median ± quartiles; from Ma et al. ) (pyramidal neuron clusters: L2/3 IT, L3–5 IT1–3, L5 ET, L5–6 NP, L6 CT, L6 IT1–2, L6B; GABAergic neuron clusters: ADARB2 KCNG1, LAMP5 LHX6 and RELN, PVALB and PVALB ChC, SST, SST HGF and NPY, VIP; non-neuronal cells: Astro, Endo, Immune, Micro, OPC, Oligo, PC, RB, SMC, VLMC) (Deseq2 test with pseudo-bulk approach for each cluster; ns, non-significant; ∗ adjusted p value < 0.05; ∗∗ adjusted p value < 0.01; ∗∗∗ adjusted p value < 0.001; ∗∗∗∗ adjusted p value < 10−4; ∗∗∗∗∗ adjusted p value < 10−6). (C) Live immunostaining for LRRC37B (see ) on HEK293T cells transfected for human, chimpanzee, and macaque LRRC37B as well as human LRRC37A2-HA (single focal plan). (D) Immunostaining after fixation and permeabilization for HA on HEK293T cells transfected for LRRC37A2-HA (single focal plan). (E) Immunofluorescent detection of LRRC37B and ankyrin-G complementary to Figure 1 H in cryosections of macaque (1–4 years old), chimpanzee (17–37 years old), and human (16–45 years old) cerebral cortex (see and Table S3).

Article Snippet: Human: HEK293T , ATCC , RRID:CVCL_1926.

Techniques: Expressing, RNA Sequencing, Immunostaining, Transfection

Journal: Cell

Article Title: LRRC37B is a human modifier of voltage-gated sodium channels and axon excitability in cortical neurons

doi: 10.1016/j.cell.2023.11.028

Figure Lengend Snippet: LRRC37 proteins are receptors for FGF13A, related to Figure 3 (A) Representative plates of ELISA-based unbiased interactome screen next to measured values. The predicted extracellular sequence of LRRC37B (LRRC37B_ECTO) fused with alkaline phosphatase (ALP) was used as a bait; using LRRC37B_ECTO-ALP immobilized in each well of 384-well plates, 920 transmembrane or secreted proteins fused to a Fc domain were used as preys in each well: among them, only FGF13A-Fc was replicated and validated as a positive hit (blue wells vs. negative wells). (B) FGF13A co-immunoprecipitates the LRRC37B extracellular part (LRRC37B_ECTO) as well as its leucine-rich repeats (LRRs) but not the extracellular part devoid of the LRR from transfected HEK293T cells; the pictures and the left and right are from different gels from the same experiment. (C) IPs of human, chimpanzee, macaque LRRC37B proteins, or human LRRC37A2 protein from HEK293T cells transfected for their cDNA and FGF13A cDNA: all LRRC37 proteins except the macaque LRRC37B binds to FGF13A. (D) IPs of LRRC37B_ECTO and other transmembrane proteins (extracellular domain fused at the N-terminal with the prolactin leader peptide and an HA tag, and at the C-terminal with the transmembrane domain of PDGF-R) from HEK293T cells transfected for their cDNA and FGF13A cDNA; stars indicate the transmembrane protein in the input; the pictures and the left and right are from different gels from the same experiment. (E) Multi-angle light scattering with size-exclusion chromatography (SEC-MALS) of LRR recombinant protein showing a primary monomeric peak, with a low oligomeric fraction. (F) Binding assay of synthetic F13ExonS-biotin to transfected LRRC37B_ECTO (n = 6 experiments), transfected LRR_ECTO (n = 3), transfected ΔLRR_ECTO (n = 3), transfected display vector (n = 6), and non-transfected cells (n = 6) HEK293T cells (fitting curves for LRRC37B_ECTO and LRR_ECTO). (G) Binding assay of synthetic F13ExonS-biotin to transfected SCN8A (Nav1.6) (n = 11 experiments), transfected empty vector (n = 9), and non-transfected cells (n = 11) HEK293T cells (fitting curves for Nav1.6).

Article Snippet: Human: HEK293T , ATCC , RRID:CVCL_1926.

Techniques: Enzyme-linked Immunosorbent Assay, Sequencing, Transfection, Multi-Angle Light Scattering, Size-exclusion Chromatography, Recombinant, Binding Assay, Plasmid Preparation

Journal: Cell

Article Title: LRRC37B is a human modifier of voltage-gated sodium channels and axon excitability in cortical neurons

doi: 10.1016/j.cell.2023.11.028

Figure Lengend Snippet: LRRC37B is a receptor for FGF13A (A) FGF13 codes for several spliced isoforms; cell lysate and medium samples of HEK293T cells transfected for FGF13A, FGF13B, FGF13VY, and FGF13-core cDNAs (stars indicate each isoform in the cell extracts) detected with FGF13 antibody. (B) LRRC37B-HA IP from HEK293T cells co-transfected for LRRC37B-HA cDNA and cDNAs coding for the different FGF13 isoforms (stars indicate each isoform in the inputs) detected with FGF13 antibody. (C) LRRC37B-HA IP from HEK293T cells transfected for LRRC37B-HA cDNA and with recombinant FGF13A or its synthetic F13ExonS applied in the culture medium detected with a FGF13A-specific antibody (used in next panels). (D and E) IP from HEK293T cells transfected with LRRC37B and mutants lacking or carrying LRR of the extracellular domain and treated with recombinant FGF13A in the culture medium (stars indicate each LRRC37B protein in the inputs). (F) Fluorescence polarization (FP) assay between LRR recombinant protein and F13ExonS peptide, F13ExonU peptide (part of FGF13B) and two random peptides (n = 9 measures; mean ± SD). (G) IP of Nav1.6 (SCN8A) from HEK293T cells transfected for SCN8A cDNA with recombinant FGF13A or its synthetic F13ExonS applied in the culture medium. (H) Nav1.6 IPs from HEK293T cells transfected for SCN8A ± FGF13A ± LRRC37B cDNAs. (I) Schematics of the LRRC37B-FGF13A-Nav1.6 interaction. See also .

Article Snippet: Human: HEK293T , ATCC , RRID:CVCL_1926.

Techniques: Transfection, Recombinant, Fluorescence, FP Assay

Journal: Cell

Article Title: LRRC37B is a human modifier of voltage-gated sodium channels and axon excitability in cortical neurons

doi: 10.1016/j.cell.2023.11.028

Figure Lengend Snippet: LRRC37B function in human neurons, related to Figures 6 and (A) LRRC37B_ECTO-Fc pull down from rat cortical extracts. AIS proteins unique for the LRRC37B_ECTO-Fc condition not found in the control condition are depicted. (B) IPs of human, chimpanzee, macaque LRRC37B proteins or human LRRC37A2 protein from HEK293T cells transfected for their cDNA and SCN1B cDNA: all LRRC37B proteins bind to SCN1B but not LRRC37A2. (C) LRRC37B, FGF13, SCN1B, and SCN8A transcripts detection in human cortical cells (from Allen Brain Map, Human MTG 10× SEA-AD dataset published in Hodge et al. ). (D) Mouse CPNs (11 neurons from 3 individuals, P28, barrel cortex) display a higher excitability than human neurons (40 neurons, from 9 individuals, from 4 to 62 years old, temporal cortex) (some data for human neurons are also included in Figure 7 E) (left, mean + SEM; right, line at median, Mann-Whitney test). (E) Electrophysiological properties complementary to Figures 7 D–7H of human neurons LRRC37B-positive (35 neurons from 12 individuals, 4–62 years old) versus LRRC37B-negative (32 neurons from 12 individuals, 4–62 years old) (lines at median; Mann-Whitney tests). (F) IV-curves (mean + SEM, ionic currents) and maximum currents (lines at median, Mann-Whitney tests) of LRRC37B-negative and LRRC37B-positive neurons. (G) Phase plot analysis of single evoked AP from LRRC37B-negative and LRRC37B-positive human neurons complementary to Figure 7 H (see in ). (H) Phase plot on single evoked AP of LRRC37B-positive and LRRC37B-negative neurons (n = 11 neurons per condition from 6 individuals, 7–56 years old; lines at median; Mann-Whitney tests) (see in ). (I) Cortical depth and soma size of the patched neurons (lines at median; Mann-Whitney tests). (J and K) AIS morphological properties of human LRRC37B-negative (110 neurons from 3 individuals, 22–38 years old) and LRRC37B-positive (93 neurons from 3 individuals, 22–38 years old) neurons (lines at median; Mann-Whitney tests). ns, non-significant; ∗ p < 0.05; ∗∗∗∗ p < 0.0001.

Article Snippet: Human: HEK293T , ATCC , RRID:CVCL_1926.

Techniques: Control, Transfection, MANN-WHITNEY

Journal: Cell

Article Title: LRRC37B is a human modifier of voltage-gated sodium channels and axon excitability in cortical neurons

doi: 10.1016/j.cell.2023.11.028

Figure Lengend Snippet: LRRC37B binds to SCN1B through its specific B domain (A and B) IP of different protein lacking or carrying the LB specific domain of the extracellular domain of LRRC37B from HEK293T cells co-transfected for the SCN1B cDNA. (C) SCN1B IPs from HEK293T cells transfected for SCN1B ± LRRC37B ± FGF13A cDNAs. (D) Nav1.6 IPs from HEK293T cells transfected for SCN8A ± SCN1B ± LRRC37B cDNAs. See also .

Article Snippet: Human: HEK293T , ATCC , RRID:CVCL_1926.

Techniques: Transfection

Journal: Cell

Article Title: LRRC37B is a human modifier of voltage-gated sodium channels and axon excitability in cortical neurons

doi: 10.1016/j.cell.2023.11.028

Figure Lengend Snippet:

Article Snippet: Human: HEK293T , ATCC , RRID:CVCL_1926.

Techniques: Recombinant, Plasmid Preparation, Magnetic Beads, Expressing, Comparison, Software