Journal: PloS one

Article Title: Cross-protective peptide vaccine against influenza A viruses developed in HLA-A*2402 human immunity model.

doi: 10.1371/journal.pone.0024626

Figure Lengend Snippet: Figure 4. An accumulation of murine CD3+ and CD8+ cells around the bronchioles in intranasally immunized mice. A24Tg mice were immunized three times at 7 to 9 days intervals i.n.(A,C,D,E) or s.c.(B) with PA130–138, PB1430–438 and PB2549–557 peptides in the presence of CpG-ODN (B,C,D), Tyrosinase206–214 plus CpG-ODN (E) or CpG-ODN plus empty-liposome solution (A). Lungs were harvested at day 7 after the final immunization, embedded in O.C.T. compound, frozen in dry ice-2-propanol. Ten mm thick frozen sections were prepared. The sections were post- fixed in acetone:ethanol (1:1) solution and blocked endogenous avidin and biotin activity, then stained with anti-mouse CD3 (A,B,C) or anti-mouse CD8a (D,E). doi:10.1371/journal.pone.0024626.g004

Article Snippet: The sections were stained with biotinylated hamster anti-mouse CD3 (eBioscience, San Diego, CA) or biotinylated rat anti-mouse CD8a (R&D Systems ) antibodies.

Techniques: Avidin-Biotin Assay, Activity Assay, Staining

Journal: PLoS ONE

Article Title: IL-23 Induces Atopic Dermatitis-Like Inflammation Instead of Psoriasis-Like Inflammation in CCR2-Deficient Mice

doi: 10.1371/journal.pone.0058196

Figure Lengend Snippet: On day 12, ( A ) mRNA of indicated cytokines was measured by real-time RT-PCR from ears of WT PBS-injected or WT and CCR2 −/− IL-23-injected mice. Average of 11 mice per genotype in 3 experiments. *p<0.01. ( B ) Intracellular cytokine staining for IL-4 on gated CD3 + CD4 + T cells following stimulation with PMA and ionomycin was performed and day 12 and analyzed by flow cytometry. Number of IL-4 + CD3 + CD4 + T cells in draining lymph node (left panel) or IL-23-injected ear (right panel). ( C ) TSLP was measured by tissue ELISA of ears isolated from PBS-injected or IL-23-injected WT and CCR2 −/− mice. Average of 3 mice per genotype. *p<0.005 CCR2 KO IL-23 vs. all other groups. ( D ) Representative immunofluorescence staining of ear skin isolated on day 12 from IL-23-injected WT C57Bl/6 or CCR2 −/− mice. Sections were stained with Ig isotype control antibody or anti-TSLP (x600, original magnification). Data are reflective of 3 mice per genotype.

Article Snippet: Sections were then washed twice in PBS 0.1% Tween and stained overnight with 1 μg of an affinity purified polyclonal antibody to mouse TSLP (R & D Systems, BAF555) diluted in PBS 0.1% saponin at 4 ° C. After washing with PBS 0.1% Tween on ice, sections were stained with 1 μg of AlexaFluor 488-conjugated donkey anti-goat IgG (Invitrogen) for 1 hour at room temperature.

Techniques: Quantitative RT-PCR, Injection, Staining, Flow Cytometry, Enzyme-linked Immunosorbent Assay, Isolation, Immunofluorescence, Control

Journal: The Journal of Clinical Investigation

Article Title: Alternative splicing of uromodulin enhances mitochondrial metabolism for adaptation to stress in kidney epithelial cells

doi: 10.1172/JCI183343

Figure Lengend Snippet: ( A ) The upper panel shows the primary structure and domains of UMOD. The 4 EGF-like domains are represented by the Roman numerals I through IV. D10C, domain with conserved 10 cysteines; ZP, zona pellucida; IHP, internal hydrophobic patch; EHP, external hydrophobic patch. The lower panel shows the exon/intron structure of the UMOD gene from Refseq (NCBI database). ( B and C ) Sashimi plot visualizes differentially spliced exons of the UMOD transcript isolated from ( B ) human and ( C ) mouse kidneys. Each numeral on the semicircle represents the number of RNA-Seq reads. Reads indicating alternative splicing sites of exon 2 and exon 10 skipping were highlighted in blue and yellow, respectively. n = 3 for human and n = 4 for mouse kidneys. ( D ) Definition of abbreviation. ( E ) Percent-splice-in (PSI) value of AS-UMOD calculated from Nanopore long-read RNA-Seq data ( n = 3 for human and n = 4 for mouse kidneys). ( F ) RT-PCR for AS-UMOD and C-UMOD from human kidney cDNA. ( G ) RT-PCR product of F was purified and subsequent Sanger sequencing confirmed the existence of AS-UMOD (exon 10 skipping UMOD ) in human kidneys. ( H ) RT-PCR for AS-Umod and C-Umod from mouse kidney cDNA. ( I ) RT-PCR product of H was purified and subsequent Sanger sequencing confirmed the existence of AS-Umod (exon 10 skipping Umod ) in mouse kidneys. Data are represented as mean ± SEM.

Article Snippet: Next, Dynabeads Biotin Binder (11047, Thermo Fisher Scientific) was conjugated with mouse anti-UMOD biotinylated antibody (BAF5175, R&D Systems).

Techniques: Isolation, RNA Sequencing, Alternative Splicing, Reverse Transcription Polymerase Chain Reaction, Purification, Sequencing

Journal: The Journal of Clinical Investigation

Article Title: Alternative splicing of uromodulin enhances mitochondrial metabolism for adaptation to stress in kidney epithelial cells

doi: 10.1172/JCI183343

Figure Lengend Snippet: ( A and B ) Relative mRNA expression of AS-Umod and C-Umod normalized to Gapdh in IRI mice. WT mice underwent sham, mild IRI, or severe IRI surgery and were harvested 24 hours after the surgery. n = 9–10 per group. ( C ) Immunofluorescence of subcortical region of murine kidneys 24 hours after the surgery. n = 5 mice per group. Scale bars: 100 μm. ( D ) Apical membrane localization of UMOD and AS-UMOD, determined by the ratio of apical membrane: whole tubules mean signal intensity was quantified using ImageJ (NIH). n = 20 tubules from 5 mild IRI kidneys for each group. ( E and F ) Relative mRNA expression of AS-Umod ( E ) and C-Umod ( F ) normalized to Gapdh in LPS-induced AKI mice. 5 mg/kg LPS was injected via intraperitoneal injection and mice were harvested 24 hours after injection. n = 6 per group. ( G and H ) Relative mRNA expression of AS-Umod ( G ) and C-Umod ( H ) normalized to Gapdh in cisplatin-induced AKI mice. 20 mg/kg cisplatin was injected via intraperitoneal injection and mice were harvested 72 hours after injection. n = 6 per group. ( I and J ) Relative mRNA expression of AS-UMOD ( I ) and C-UMOD ( J ) normalized to NKCC2 in human kidney samples from the KPMP. n = 7–12 per group. ( K ) Relative mRNA expression of AS-Umod and C-Umod normalized to Gapdh in MKTAL cells treated with various concentrations of hydrogen peroxide (H 2 O 2 ) for 6 hours. n = 4 per group. ( L ) Relative mRNA expression of AS-Umod , C-Umod , and Glut1 normalized to Hprt in hypoxia conditions. MKTAL cells were cultured in control (normoxia) or hypoxia conditions for 6 hours. n = 4 per group. Data were analyzed by unpaired t test (between 2 conditions, D – H , and L ) or 1-way ANOVA with embedded comparisons between 2 individual groups (among multiple conditions, A , B , and I – K ) and are represented as mean ± SEM. * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001.

Article Snippet: Next, Dynabeads Biotin Binder (11047, Thermo Fisher Scientific) was conjugated with mouse anti-UMOD biotinylated antibody (BAF5175, R&D Systems).

Techniques: Expressing, Immunofluorescence, Membrane, Injection, Cell Culture, Control

Journal: The Journal of Clinical Investigation

Article Title: Alternative splicing of uromodulin enhances mitochondrial metabolism for adaptation to stress in kidney epithelial cells

doi: 10.1172/JCI183343

Figure Lengend Snippet: MDCK cells stably expressing C-UMOD or AS-UMOD were established by lentiviral transduction. ( A and B ) Immunoblotting of UMOD in MDCK cell lysate and medium, respectively. Coomassie staining was used as a loading control for medium. n = 4. ( C ) Immunofluorescence of C-UMOD and AS-UMOD in MDCK cells. n = 3. Scale bar: 10 μm. ( D ) Immunofluorescence of UMOD C148W, an ADTKD-causing mutant in MDCK cells. n = 3. Scale bar: 10 μm. ( E ) Relative mRNA expression of ER stress–related genes normalized to GAPDH expression. n = 3. ( F ) LDH assay in MDCK cells. MDCK cells were cultured in normoxia or hypoxia conditions for 6 hours. LDH concentration in the media was measured and normalized to total cell number. n = 3. Data were analyzed by unpaired t test (between 2 conditions, B ) or 1-way ANOVA with embedded comparisons between 2 individual groups (among multiple conditions, E and F ) and are represented as mean ± SEM. * P < 0.05; *** P < 0.001; **** P < 0.0001.

Article Snippet: Next, Dynabeads Biotin Binder (11047, Thermo Fisher Scientific) was conjugated with mouse anti-UMOD biotinylated antibody (BAF5175, R&D Systems).

Techniques: Stable Transfection, Expressing, Transduction, Western Blot, Staining, Control, Immunofluorescence, Mutagenesis, Lactate Dehydrogenase Assay, Cell Culture, Concentration Assay

Journal: The Journal of Clinical Investigation

Article Title: Alternative splicing of uromodulin enhances mitochondrial metabolism for adaptation to stress in kidney epithelial cells

doi: 10.1172/JCI183343

Figure Lengend Snippet: ( A ) Immunoblotting of MDCK cells expressing C-UMOD or AS-UMOD after subcellular fractionation. The same amount of protein was applied for each fraction. The ratio of mitochondrial/total (mitochondrial, cytosolic, and membrane) UMOD expression was quantified by densitometry analysis. n = 3. ( B ) Immunofluorescence of MDCK cells expressing C-UMOD or AS-UMOD. Colocalization analysis between UMOD and mitochondria (Mitotracker) in MDCK cells. Manders’ tM1 represents a fraction of UMOD overlapping with mitochondria. n = 30 cells per group from 3 independent experiments. Scale bar: 10 μm. ( C ) ATP/ADP ratio of mitochondria isolated from MDCK cells. n = 4. ( D ) Mitochondrial respiration measurement in MDCK cells expressing C-UMOD or AS-UMOD using Seahorse. OCR, oxygen consumption rate; FCCP, carbonyl cyanide p -trifluoromethoxyphenylhydrazone. n = 3. ( E ) Transmission electron microscopy in MDCK cells expressing C-UMOD or AS-UMOD. Scale bar: 1 μm. Mitochondrial number per 100 μm 2 cell area (excluding nucleus) was quantitated. n = 18 cells for each group from 2 independent experiments. ( F ) Relative mRNA expression of PGC1 α and NRF1 normalized to GAPDH . n = 4. ( G ) Interactome map of C-UMOD and AS-UMOD in MDCK cells obtained from AP-MS analysis. ( H ) Coimmunoprecipitation with anti-UMOD antibody to validate the AP-MS analysis. Asterisk indicates lower band is the target band for SLC25A22. n = 2. ( I ) The ratio of mitochondrial/cytosolic glutamate levels in MDCK cells. n = 3. ( J ) NAD + levels normalized to protein concentration. n = 3. ( K ) ADP/ATP carrier-mediated ATP export after ADP addition to the isolated mitochondria from MDCK cells. n = 3. Data were analyzed by unpaired t test (between 2 conditions, A , B , D – F , and I – K ) or 1-way ANOVA with embedded comparisons between 2 individual groups (among multiple conditions, C ) and are represented as mean ± SEM. * P < 0.05; ** P < 0.01; **** P < 0.0001.

Article Snippet: Next, Dynabeads Biotin Binder (11047, Thermo Fisher Scientific) was conjugated with mouse anti-UMOD biotinylated antibody (BAF5175, R&D Systems).

Techniques: Western Blot, Expressing, Fractionation, Membrane, Immunofluorescence, Isolation, Transmission Assay, Electron Microscopy, Protein-Protein interactions, Protein Concentration

Journal: The Journal of Clinical Investigation

Article Title: Alternative splicing of uromodulin enhances mitochondrial metabolism for adaptation to stress in kidney epithelial cells

doi: 10.1172/JCI183343

Figure Lengend Snippet: ( A ) Two sgRNAs were designed to cut the intronic region around exon 10. ( B and C ) Genotyping PCR ( B ) and subsequent Sanger sequencing of the PCR product ( C ) confirmed successful heterozygous knockout of Umod exon 10. ( D ) Relative mRNA expression of AS-Umod and C-Umod normalized to Gapdh in WT (Exon10 +/+ ) and Umod exon10 heterozygous knockout (Exon10 +/– ) MKTAL cells. n = 4. ( E and F ) Immunoblotting of UMOD in cell lysate ( E ) and medium ( F ). Red asterisk corresponds to AS-UMOD. Coomassie staining was used as a loading control for medium. n = 3. ( G ) NAD + levels normalized to protein concentration. n = 4. ( H ) ATP levels normalized to protein concentration. n = 4. ( I ) ATP/ADP ratio of mitochondria isolated from Exon 10 +/+ and Exon 10 +/– MKTAL cells. n = 4. Data were analyzed by unpaired t test and are represented as mean ± SEM. * P < 0.05; ** P < 0.01; *** P < 0.001.

Article Snippet: Next, Dynabeads Biotin Binder (11047, Thermo Fisher Scientific) was conjugated with mouse anti-UMOD biotinylated antibody (BAF5175, R&D Systems).

Techniques: Sequencing, Knock-Out, Expressing, Western Blot, Staining, Control, Protein Concentration, Isolation

Journal: The Journal of Clinical Investigation

Article Title: Alternative splicing of uromodulin enhances mitochondrial metabolism for adaptation to stress in kidney epithelial cells

doi: 10.1172/JCI183343

Figure Lengend Snippet: HA-C-UMOD and Myc-AS-UMOD were cotransduced to MDCK cells and their interaction was evaluated. ( A ) Coimmunoprecipitation with anti-HA antibody in MDCK cells expressing MYC-AS-UMOD only (lane 1) or an equal amount of HA-C-UMOD and Myc-AS-UMOD (lane 2). CANX was used as a positive control of coimmunoprecipitation. Red asterisk corresponds to CANX. n = 2. ( B ) Intracellular ATP levels normalized to protein concentration. n = 4. ( C and D ) Immunoblotting analysis of MDCK cells expressing HA-C-UMOD only (lane 1) or equal amount of HA-C-UMOD and Myc-AS-UMOD (lane 2). Densitometric analysis of HA-C-UMOD is presented. n = 3. ( E ) Secreted HA-C-UMOD normalized by intracellular HA-C-UMOD expression. n = 3. Data were analyzed by unpaired t test (between 2 conditions, C – E ) or 1-way ANOVA with embedded comparisons between 2 individual groups (among multiple conditions, B ) and are represented as mean ± SEM. * P < 0.05.

Article Snippet: Next, Dynabeads Biotin Binder (11047, Thermo Fisher Scientific) was conjugated with mouse anti-UMOD biotinylated antibody (BAF5175, R&D Systems).

Techniques: Expressing, Positive Control, Protein Concentration, Western Blot

Journal: The Journal of Clinical Investigation

Article Title: Alternative splicing of uromodulin enhances mitochondrial metabolism for adaptation to stress in kidney epithelial cells

doi: 10.1172/JCI183343

Figure Lengend Snippet: ( A ) Design of SSOs to induce AS-Umod expression. Numbers in parentheses indicate position from the first base of exon 10. ( B ) Relative mRNA expression of AS-Umod normalized to Gapdh in MKTAL cells transfected with 30nM SSOs for 24 hours. Lipofectamine alone and nontargeted SSO were used as negative controls. n = 3. ( C ) Relative mRNA expression of AS-Umod and C-Umod normalized to Gapdh in MKTAL cells transfected with various concentrations of Umod SSO for 48 hours. Umod SSO corresponds to SSO (–13). n = 3. ( D – I ) MKTAL cells were treated with 30 nM scrambled SSO or Umod SSO for 48 hours. ( D and E ) Immunoblotting of UMOD in cell lysate and medium, respectively. Red asterisk corresponds to AS-UMOD. Coomassie staining was used as a loading control for medium. n = 4. ( F ) Immunofluorescence of UMOD. n = 2. Scale bar: 10 μm. ( G ) The ratio of mitochondrial/cytosolic glutamate levels. n = 3. ( H ) NAD + levels normalized to protein concentration. n = 3. ( I ) ATP levels normalized to protein concentration. n = 3. Data were analyzed by unpaired t test (between 2 conditions, E and G – I ) or 1-way ANOVA with embedded comparisons between 2 individual groups (among multiple conditions, C ) and are represented as mean ± SEM. * P < 0.05; ** P < 0.01; **** P < 0.0001.

Article Snippet: Next, Dynabeads Biotin Binder (11047, Thermo Fisher Scientific) was conjugated with mouse anti-UMOD biotinylated antibody (BAF5175, R&D Systems).

Techniques: Expressing, Transfection, Western Blot, Staining, Control, Immunofluorescence, Protein Concentration

Journal: The Journal of Clinical Investigation

Article Title: Alternative splicing of uromodulin enhances mitochondrial metabolism for adaptation to stress in kidney epithelial cells

doi: 10.1172/JCI183343

Figure Lengend Snippet: WT mice underwent severe IRI and SSO treatment (25 mg/kg) and were harvested 72 hours after IRI. ( A ) Schematic of experimental design. ( B ) Relative mRNA expression of AS-Umod and C-Umod normalized to Gapdh . n = 10–11 per group. ( C ) Immunofluorescence of murine kidneys. White arrows indicate AS-UMOD, which is induced in the cytosol of TAL cells after Umod SSO treatment. n = 4 mice per group. Scale bars: 50 μm. ( D ) Serum creatinine and urea concentration. n = 14–16 per group. ( E ) PAS-stained kidney sections and quantification of injury. n = 9–11 per group. Scale bar: 500 μm. ( F ) Relative mRNA expression of injury-related genes normalized to Gapdh in the whole kidney. n = 10–11 per group. ( G and H ) Primary TAL cells were isolated by magnetic cell separation, and cells unbound to the beads were defined as non-TAL cells. ( G ) Relative mRNA expression of injury-related genes normalized to Gapdh in TAL and non-TAL cells. n = 7–8 per group. ( H ) ATP levels normalized to protein concentration in TAL and non-TAL cells. n = 7–8 per group. Data were analyzed by unpaired t test and are represented as mean ± SEM. * P < 0.05; **** P < 0.0001.

Article Snippet: Next, Dynabeads Biotin Binder (11047, Thermo Fisher Scientific) was conjugated with mouse anti-UMOD biotinylated antibody (BAF5175, R&D Systems).

Techniques: Expressing, Immunofluorescence, Concentration Assay, Staining, Isolation, Magnetic Cell Separation, Protein Concentration

Journal: The Journal of Clinical Investigation

Article Title: Alternative splicing of uromodulin enhances mitochondrial metabolism for adaptation to stress in kidney epithelial cells

doi: 10.1172/JCI183343

Figure Lengend Snippet: C-UMOD is a GPI-anchored protein and is sorted to the plasma membrane. C-UMOD regulates the activities of membrane transporters and maintains extracellular homeostasis once secreted into the extracellular region. AKI induces alternative splicing of UMOD and generates AS-UMOD, a non-GPI anchored isoform. AS-UMOD showed preferential localization in the mitochondria compared with C-UMOD, facilitating mitochondrial energy generation as a metabolic adaptation to cellular injury. However, mitochondrial localization of AS-UMOD remains partial, and we cannot exclude the possibility that AS-UMOD in ER could also affect mitochondrial function. The mechanism by which a portion of AS-UMOD targets the mitochondria remains unknown. The schema was created in BioRender. Nanamatsu, A. (2025) https://BioRender.com/k97g401

Article Snippet: Next, Dynabeads Biotin Binder (11047, Thermo Fisher Scientific) was conjugated with mouse anti-UMOD biotinylated antibody (BAF5175, R&D Systems).

Techniques: Clinical Proteomics, Membrane, Alternative Splicing

Journal: JCI insight

Article Title: Collectin-11 promotes cancer cell proliferation and tumor growth.

doi: 10.1172/jci.insight.159452

Figure Lengend Snippet: Figure 3. Colec11–/– mice exhibit less immunosuppressive TME. Tumors excised from Colec11+/+ (WT) or Colec11–/– (KO) mice (d14) were used for analyzing TME. (A–C) Tumor infiltrates analyzed by flow cytometry. (A) CD45+ cells. (B) Subsets of tumor-infiltrating leukocytes analyzed by flow cytometry. Data were analyzed by unpaired t test (n = 18 mice per group, pooled from 4 experiments). Each dot represents an individual mouse. (C) A bar chat representing proportion of subsets in CD45+ cells shown in B. (D) Representative microscopy images of immunochemical staining for CD11b (green)/CD3 (red)/DAPI (blue) and F4/80 (green)/CD3 (red)/DAPI (blue). Scale bar: 50 μm. (E) Representative microscopy images of immunochemical staining for CD8 (red)/DAPI (blue) in tumor edge and core areas. Scale bar: 50 μm. (F). qPCR analysis in tumor tissues. Data were analyzed by unpaired t test (n = 8 mice per group). *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.

Article Snippet: The following antibodies were used in immunochemical staining: monoclonal rat anti–mouse CD45 (103120), CD11b (101202), and F4/80 (123102) (all from BioLegend); rat anti–mouse CD31(557355, BD Biosciences); rabbit anti-mouse CD3 (ab237721), CD8α (ab217344), VWF (ab6994), and rabbit anti– human COLEC11 (ab238585) (all from Abcam); rabbit anti–mouse H2-Ab1 (A18658, ABclonal); rat anti– mouse CD68 (FA11) (Bio-Rad); goat anti–mouse CD206(AF2535) (from R&D systems); rabbit anti–mouse Ki67 (9129, Cell Signaling Technology); Alexa Fluor 488 goat anti–rat IgG (catalog 405418), Alexa Fluor 555 goat anti–rat IgG (catalog 405420), and Alexa Fluor 647 donkey anti–rabbit IgG (catalog 406414) (all from BioLegend); and Alexa Fluor 488 goat anti–rabbit IgG (catalog 4412) and Alexa Flour 594 goat anti– rabbit IgG (catalog 8889) (both from Cell Signaling Technology).

Techniques: Flow Cytometry, Microscopy, Staining

Journal: bioRxiv

Article Title: The late onset Alzheimer’s disease risk factor iRhom2/RHBDF2 is a modifier of microglial TREM2 proteolysis

doi: 10.1101/2024.09.13.612888

Figure Lengend Snippet: A-B. Proteomic hiSPECS analysis comparing BV2 iRhom2 -/- (A) or ADAM17 -/- (B) microglia to BV2 NTC microglia. Volcano plots depict the log2 abundance change in the secretome and the negative log10 p-value for each protein (two-sample t-test, N = 12). The permutation-based false discovery rate estimation is represented by the black hyperbolic dashed line (FDR, p = 0.05, s0 = 0.1). Vertical dotted lines indicate changes of larger or smaller than two-fold in the log scale. Proteins with a p-value below 0.05 are highlighted with red (FDR significant) or black circles. For ease of visualization, the secretome data of the two iRhom2 (iR2 -/- : KO #1 and #2) and ADAM17 (A17 -/- : KO #1 and #2) clones were pooled and compared to the pooled secretome of the two NTC clones (NTC #1 and NTC #2) non-targeting control clones. C. The peptide distribution according to the specific protein domains of the indicated iRhom2/ADAM17 substrates was visualized using the web tool Quantitative Analysis of Regulated Intramembrane Proteolysis (QARIP) ( Ivankov et al , 2013 ). D. Validation of iRhom2/ADAM17-mediated TREM2 ectodomain shedding in BV2 cells. The supernatant prepared for the hiSPECS analysis in (A and B) was subjected to ELISA. sTREM2 levels were quantified in conditioned media of the indicated BV2 KO cell lines (N ≥5). NTC (N = 10) contains the combined data of NTC #1 (N = 5) and NTC #2 (N = 5) clones. NTC was used as baseline, and its average normalized to 100. One-way ANOVA with Tukey’s correction for multiple comparisons. E. Immunoblot analysis of TREM2 in BV2 KO cell lines. Highly glycosylated, mature species of TREM2 were enriched in the ADAM17 and iRhom2 KO lines. Calnexin served as loading control. F. Surface abundance of TREM2 in BV2 KO cells. Cells were labeled with an antibody against TREM2 or isotype control to assess the geometric mean intensity of indicated BV2 KO cell lines using flow cytometry (N ≥ 3). NTC (N = 6) contains the combined data of NTC #1 (N = 3) and NTC #2 (N = 3) clones. BV2 NTC was used as baseline, and its average normalized to 100. One-way ANOVA with Tukey’s correction for multiple comparisons. Data information: Data (D, F) are represented as means ± SD from at least three independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001.

Article Snippet: After discarding the blocking buffer, plates were incubated with 25 μl of a biotinylated anti-TREM2 antibody (R&D systems, BAF1729) at 0.125 μg/ml for 90 minutes at room temperature.

Techniques: Clone Assay, Control, Biomarker Discovery, Enzyme-linked Immunosorbent Assay, Western Blot, Labeling, Flow Cytometry

Journal: bioRxiv

Article Title: The late onset Alzheimer’s disease risk factor iRhom2/RHBDF2 is a modifier of microglial TREM2 proteolysis

doi: 10.1101/2024.09.13.612888

Figure Lengend Snippet: A. Proteomic hiSPECS analysis comparing the secretome of primary microglia isolated from wild-type (WT) or iRhom2 -/- (iR2 KO) pups. The volcano plot displays protein abundance changes between WT and iR2 KO supernatants. The log2 fold change is plotted against the p-value (-log10). The permutation-based false discovery rate estimation is represented by the black hyperbolic dashed line (FDR, p = 0.05, s0 = 0.1). Proteins with a p-value below 0.05 are highlighted with red (FDR significant) or black circles. Vertical dotted lines indicate changes of larger or smaller than two-fold in the log scale. B. The peptide distribution according to the specific protein domains of the indicated iRhom2/ADAM17 substrates was visualized using QARIP (see ). C. Validation of iRhom2/ADAM17-mediated TREM2 ectodomain shedding in primary iRhom2 -/- microglia. The supernatant prepared for the hiSPECS analysis in (A) was subjected to ELISA. sTREM2 levels were quantified in supernatants of primary microglia (N ≥ 7). Two-sided independent Student’s t-test was performed. D. Immunoblot analysis of TREM2 levels in primary microglia lysates derived from wild-type or iRhom2 -/- pups. Three biological replicates of either genotype are shown. Highly glycosylated, mature species of TREM2 were enriched in the lysates of iRhom2-deficient microglia. Actin served as loading control. Data information: Data (C) are represented as means ± SD from at least three independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001.

Article Snippet: After discarding the blocking buffer, plates were incubated with 25 μl of a biotinylated anti-TREM2 antibody (R&D systems, BAF1729) at 0.125 μg/ml for 90 minutes at room temperature.

Techniques: Isolation, Quantitative Proteomics, Biomarker Discovery, Enzyme-linked Immunosorbent Assay, Western Blot, Derivative Assay, Control

Journal: bioRxiv

Article Title: The late onset Alzheimer’s disease risk factor iRhom2/RHBDF2 is a modifier of microglial TREM2 proteolysis

doi: 10.1101/2024.09.13.612888

Figure Lengend Snippet: A. Immunoblot analysis of TREM2 signaling in cell lysates isolated from wild-type (WT) or iRhom2 -/- (iR2 KO) BMDMs. Three biological replicates of either genotype are shown. The membranes were probed with antibodies against iRhom2, ADAM17, TREM2, or antibodies against SYK or phospho-SYK-Y519/520. Calnexin and Actin served as loading control. B. Quantification of phospho-SYK-Y519/520 levels in immunoblots as shown in (A). iRhom2 -/- BMDMs showed stronger SYK phosphorylation than wild-type BMDMs (N = 6). The phospho-SYK/total-SYK ratio was normalized to wild-type. Two-sided independent Student’s t-test was performed. C. iRhom2/ADAM17-mediated TREM2 ectodomain shedding in iRhom2 -/- BMDMs. The sTREM2 levels were quantified in supernatants from cultures in (A) by ELISA (N = 6). Two-sided independent Student’s t-test. Data information: Data (B, C) are represented as means ± SD from three independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001.

Article Snippet: After discarding the blocking buffer, plates were incubated with 25 μl of a biotinylated anti-TREM2 antibody (R&D systems, BAF1729) at 0.125 μg/ml for 90 minutes at room temperature.

Techniques: Western Blot, Isolation, Control, Phospho-proteomics, Enzyme-linked Immunosorbent Assay

Journal: Gut

Article Title: SR-PSOX/CXCL16 plays a critical role in the progression of colonic inflammation.

doi: 10.1136/gut.2010.221879

Figure Lengend Snippet: Figure 2 SR-PSOX/CXCL16 plays a role in phagocytosis of bacterial components and the production of interleukin 12 (IL-12) by macrophages. Thioglycollate-elicited peritoneal macrophages from SR-PSOX/CXCL16 knockout (KO) and wild-type (WT) mice were subjected to an ex vivo phagocytosis assay against bacteria. (A) Microscopic observation of phagocytosis was performed in peritoneal macrophages from WT mice (upper column) and SR-PSOX/CXCL16 KO mice (lower column). The upper and lower panels of each column show light microscopic findings and fluorescent images, respectively. Scale bars, 25 mm. (B) Fluorescence intensity, from which the fluorescence values of the no-cell background control wells were subtracted, was measured using a microplate reader at the indicated times. The statistical comparison of fluorescence intensity between WT (open circles) and SR-PSOX/CXCL16 KO macrophages (filled circles) was assessed by repeated measure analysis of variance followed by unpaired Student t test. (C) Peritoneal macrophages from WT (open bars) and SR-PSOX/CXCL16 KO mice (filled bars) were incubated with 500 U/ml interferon g (IFNg) for 16 h, followed by stimulation with 100 ng/ml lipopolysaccharide (LPS) or 30 mg caecal bacterial lysate (CBL) for 24 h, and the culture supernatants were analysed by ELISA to measure the concentrations of IL-6 and IL-12/23 p40. The results are expressed as means6SEM of the data from three independent experiments. The statistical difference was determined by unpaired Student t test. *p<0.05 and **p<0.01 between SR-PSOX/ CXCL16 KO and WT macrophages.

Article Snippet: The sections were incubated with fluorescein isothiocyanate (FITC)-conjugated anti-CD11b mAb (1:200; BD Pharmingen) and biotinylated anti-CXCL16 antibody (1:1000; R&D Systems) or goat IgG isotype control overnight at 48C.

Techniques: Knock-Out, Ex Vivo, Phagocytosis Assay, Bacteria, Fluorescence, Control, Comparison, Incubation, Enzyme-linked Immunosorbent Assay

Journal: Gut

Article Title: SR-PSOX/CXCL16 plays a critical role in the progression of colonic inflammation.

doi: 10.1136/gut.2010.221879

Figure Lengend Snippet: Figure 3 SR-PSOX/CXCL16 levels are higher in mice with dextran sulfate sodium (DSS)-induced colitis. (A) The serum levels of SR-PSOX/ CXCL16 in mice with DSS-induced colitis on day 8 and control mice were measured by ELISA. (B). The gene expression of SR-PSOX/CXCL16 in colonic tissues with or without DSS-induced colitis was determined by quantitative real-time reverse transcriptionePCR (RTePCR) and was normalised to glyceraldehyde phosphate dehydrogenase (GAPDH). (C). The production of SR-PSOX/CXCL16 in colonic tissues with or without DSS-induced colitis was investigated by western blot analysis. (D) SR- PSOX/CXCL16 concentrations in supernatants of colon fragment cultures were measured by ELISA. The results are expressed as means6SEM (n¼10 in each group). (A), (B) and (D) The statistical difference was determined by unpaired Student t test. *p<0.05 and **p<0.01 between mice with DSS-induced colitis and normal controls.

Article Snippet: The sections were incubated with fluorescein isothiocyanate (FITC)-conjugated anti-CD11b mAb (1:200; BD Pharmingen) and biotinylated anti-CXCL16 antibody (1:1000; R&D Systems) or goat IgG isotype control overnight at 48C.

Techniques: Control, Enzyme-linked Immunosorbent Assay, Gene Expression, Western Blot

Journal: Gut

Article Title: SR-PSOX/CXCL16 plays a critical role in the progression of colonic inflammation.

doi: 10.1136/gut.2010.221879

Figure Lengend Snippet: Figure 4 SR-PSOX/CXCL16 is expressed predominantly on macrophages in colonic tissues of mice with dextran sulfate sodium (DSS)-induced colitis. (A) Immunostaining was performed in Peyer’s patches as a positive control, normal colons and colons with 3% DSS-induced colitis. Serial sections of each tissue were stained with H&E, control goat immunoglobulin G (IgG) and anti-mouse SR-PSOX/CXCL16 monoclonal antibody. (B). Immunofluorescent staining was performed in normal colons and colons with 3% DSS-induced colitis using antibodies against CD11b (green), SR-PSOX/CXCL16 (red) and control goat IgG. The merged images and their magnified images are shown. Scale bars, 50 mm (A), 20 mm (B, left 3 lanes) and 10 mm (B, right lane).

Article Snippet: The sections were incubated with fluorescein isothiocyanate (FITC)-conjugated anti-CD11b mAb (1:200; BD Pharmingen) and biotinylated anti-CXCL16 antibody (1:1000; R&D Systems) or goat IgG isotype control overnight at 48C.

Techniques: Immunostaining, Positive Control, Staining, Control

Journal: Gut

Article Title: SR-PSOX/CXCL16 plays a critical role in the progression of colonic inflammation.

doi: 10.1136/gut.2010.221879

Figure Lengend Snippet: Figure 5 Activity of dextran sulfate sodium (DSS)-induced colitis is lower in SR-PSOX/CXCL16 knockout (KO) mice. (A) Serial change in body weight in SR-PSOX/CXCL16 KO and wild-type (WT) mice with or without 3% DSS-induced colitis. Data are expressed as the percentage change from the starting body weight. (B) Representative image and colonic length in SR-PSOX/CXCL16 KO and WT mice with or without 3% DSS-induced colitis on day 8. (C) Representative histological findings and the scores of colonic inflammation of SR-PSOX/CXCL16 KO and WT mice withor without 3% DSS-induced colitis on day 8. Scale bars, 100 mm. (D) Colonic tissues of SR-PSOX/CXCL16 KO and WT mice with or without 3% DSS-induced colitis were incubated with fluorescein isothiocyanate (FITC)-conjugated CD3, CD11b and CD11c, followed by nuclear counterstaining with 4‘,6-diamidino-2-phenylindole (DAPI). Scale bars, 100 mm. (E) Fluorescent in situ hybridization analysis was performed with colonic tissues of SR-PSOX/CXCL16 KO and WT mice with or without 3% DSS-induced colitis using eubacterial oligonucleotide probe EUB-338 (red), followed by DAPI (blue). Scale bars, 50 mm (left two lanes) and 20 mm (right lane). (F) Colonic macrophages from SR-PSOX/CXCL16 KO and WT mice were subjected to an ex vivo phagocytosis assay against bacteria. Scale bars, 25 mm. (G) MLN cells from SR-PSOX/CXCL16 KO and WT mice on days 0, 5 and 8 after administration of 3% DSS were cultured with immobilised anti-CD3 plus CD28. Supernatants were collected after 72 h and subjected to ELISA to measure the concentration of interferon g (IFNg) and interleukin 17 (IL-17). (A)e(D), (F) and (G) The results are expressed as means6SEM (n¼10e12 in each group). The statistical comparison was assessed by repeated measure analysis of variance followed by unpaired Student t test (A) and (F). The statistical difference was determined by unpaired Student t test (B), (D) and (G) or ManneWhitney U test (C). *p<0.05 and **p<0.01 between SR-PSOX/CXCL16 KO mice and WT mice with DSS-induced colitis.

Article Snippet: The sections were incubated with fluorescein isothiocyanate (FITC)-conjugated anti-CD11b mAb (1:200; BD Pharmingen) and biotinylated anti-CXCL16 antibody (1:1000; R&D Systems) or goat IgG isotype control overnight at 48C.

Techniques: Activity Assay, Knock-Out, Incubation, In Situ Hybridization, Ex Vivo, Phagocytosis Assay, Bacteria, Cell Culture, Enzyme-linked Immunosorbent Assay, Concentration Assay, Comparison

Journal: Protein engineering, design & selection : PEDS

Article Title: Engineering a pharmacologically superior form of leptin for the treatment of obesity.

doi: 10.1093/protein/gzh102

Figure Lengend Snippet: Fig. 1. Transient expression analysis of leptin fusion proteins. (a) Expression vectors for (1) muFc (control), (2) muFc–muLeptin, (3) muLeptin–muFc and (4) muLeptin linker-muFc were used to transfect 293 cells. Total cell lysates (C) and supernatants (S) were analyzed by western blot analysis using horseradish peroxidase (HRP)-conjugated anti-mouse Fc-g antisera (upper gel) and biotinylated anti-mouse leptin antibody (R&D Systems) (lower gel). Under reducing conditions, muFc and muFc–muLeptin have apparent molecular weights of 33 and 48 kDa, respectively. (b) Non-reducing SDS-PAGE comparison of the soluble fractions obtained from the expression of (1) muFc–muLeptin, (2) huFcg1–huLeptin, (3) huFcg2h–huLeptin and (4) huFcg2–huLeptin. The misfolding and aggregation of the huFcg2–huLeptin can clearly be seen, resulting from intermolecular cross-linking of the four disulfide bridges present in the human Fcg2 domain. By contrast, the modified Fcg2h variant with only two disulfide bridges shows the uniform and high-yield expression of a single molecular species.

Article Snippet: Total cell lysates (C) and supernatants (S) were analyzed by western blot analysis using horseradish peroxidase (HRP)-conjugated anti-mouse Fc-g antisera (upper gel) and biotinylated anti-mouse leptin antibody (R&D Systems) (lower gel).

Techniques: Expressing, Control, Western Blot, SDS Page, Comparison, Modification, Variant Assay

Journal: Journal of Clinical Investigation

Article Title: Monocytes and interstitial macrophages contribute to hypoxic pulmonary hypertension

doi: 10.1172/jci176865

Figure Lengend Snippet: Figure 1. Exposure to high altitude results in PH and increased secretion of inflammatory classical monocyte ligands from the lungs. (A) Schematic showing hypoxia exposure time course in wildtype mice. Duration of hypoxia exposure is directly proportional to (B) RVSP and RV hypertrophy as measured by Fulton Index (N=6-13/group). At 3 days of hypoxia, increased protein expression of classical monocyte ligands (C) CCL2 (N=6-11/group) and (D) CCL12 (N=6- 11/group), whereas significantly lower levels of nonclassical monocyte ligand (E) CX3CL1 (N=6/group) in the lungs. (F) Higher CCL2 gradient in lungs and in the (G) peripheral blood of wildtype mice following 3 days of hypoxia exposure (N=5/group). Data in all panels were obtained from female mice. Statistical analysis was conducted using ANOVA, followed by Tukey's post hoc test. *P<0.05, **P<0.01, ****P<0.0001. N=number of animals, mean±SD, CI=confidence interval.

Article Snippet: Neutralizing Antibody and Pharmacological Treatment: Neutralizing mouse antibodies against CCL2 (Clone: 2H5; Cat.# BE0185), CCL7 (R&D System; Cat.# AF-456-NA), and isotype control (Cat.# BE0091; BioXCell, West Lebanon, NH, USA) were reconstituted in phosphatebuffered saline (PBS).

Techniques: Expressing

Journal: Journal of Clinical Investigation

Article Title: Monocytes and interstitial macrophages contribute to hypoxic pulmonary hypertension

doi: 10.1172/jci176865

Figure Lengend Snippet: Figure 4: Genetic and pharmacologic blockade of CCR2-CCL2 axis protects from hypoxic PH. (A) Schematic showing the BM reconstitution of Ccr2-/- and WT BM into lethally irradiated wildtype mice. Wildtype mice reconstituted with Ccr2-/- BM were protected from hypoxic PH by attenuated (B) RVSP (N=7-11/group) and (C) RV hypertrophy (N=7-11/group) as measured by Fulton Index, compared to wildtype mice that were reconstituted with wildtype BM. (D) Schematic showing pharmacological blockade of CCR2 ligands CCL2 or CCL7 using anti-CCL2 or anti-CCL7 neutralizing antibody treatment. Hypoxia exposed wildtype mice treated with CCL2 NAb but not CCL7 NAb showed lower (E) RVSP (N=6/group) and (F) RV hypertrophy (N=6/group). TSP-1 levels in (G) lungs (N=6/group) and (H) blood (N=6/group); and TGF-β1 levels in (I) lungs (N=6/group) and (J) blood (N=6/group) compared to wildtype mice treated with isotype control antibody. Data in all panels followed a normal distribution. ANOVA with the Tukey test was performed for multiple comparisons. Data were obtained from the female mice. mean ± SD

Article Snippet: Neutralizing Antibody and Pharmacological Treatment: Neutralizing mouse antibodies against CCL2 (Clone: 2H5; Cat.# BE0185), CCL7 (R&D System; Cat.# AF-456-NA), and isotype control (Cat.# BE0091; BioXCell, West Lebanon, NH, USA) were reconstituted in phosphatebuffered saline (PBS).

Techniques: Irradiation, Control

Journal: Journal of Clinical Investigation

Article Title: Monocytes and interstitial macrophages contribute to hypoxic pulmonary hypertension

doi: 10.1172/jci176865

Figure Lengend Snippet: Figure 5: Resident IMs are a major source of CCL2 and recruited IMs are a major source of pathologic TSP-1 in hypoxic PH. (A) Flow cytometry analysis using Ccl2RFP-flox reporter mice showed a higher number of CCL2+ IMs (N=14/group; N=14/group, 9F and 5M in Nx; 8F and 6M in Hx), and (B) FOLR2+ IMs are a major source of CCL2 (N=14/group). (C) Hypoxia exposed wildtype mice following intracellular CCL2 staining by flow cytometry also showed a higher number of CCL2+ IMs (N=7/group, female mice). (D). IM subpopulation analysis using flow

Article Snippet: Neutralizing Antibody and Pharmacological Treatment: Neutralizing mouse antibodies against CCL2 (Clone: 2H5; Cat.# BE0185), CCL7 (R&D System; Cat.# AF-456-NA), and isotype control (Cat.# BE0091; BioXCell, West Lebanon, NH, USA) were reconstituted in phosphatebuffered saline (PBS).

Techniques: Flow Cytometry, Staining

Journal: Journal of Clinical Investigation

Article Title: Monocytes and interstitial macrophages contribute to hypoxic pulmonary hypertension

doi: 10.1172/jci176865

Figure Lengend Snippet: Figure 8: DEX prophylaxis blunts CCL2 production by resident IMs and blocks the recruitment of TSP-1 producing CCR2+ IMs in hypoxia. (A) DEX prophylactically-treated, hypoxia-exposed Ccl2RFP-flox reporter mice exhibited a significant reduction in CCL2+ IMs, particularly in (B) CCL2RFP+ resident IMs (N=7/group). Additionally, (C) intracellular CCL2 flow cytometry analysis in DEX prophylactically-treated hypoxia-exposed wildtype mice revealed a

Article Snippet: Neutralizing Antibody and Pharmacological Treatment: Neutralizing mouse antibodies against CCL2 (Clone: 2H5; Cat.# BE0185), CCL7 (R&D System; Cat.# AF-456-NA), and isotype control (Cat.# BE0091; BioXCell, West Lebanon, NH, USA) were reconstituted in phosphatebuffered saline (PBS).

Techniques: Flow Cytometry