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: Journal of colloid and interface science

Article Title: Targeting cancer cell adhesion molecule, CD146, with low-dose gold nanorods and mild hyperthermia disrupts actin cytoskeleton and cancer cell migration.

doi: 10.1016/j.jcis.2021.05.144

Figure Lengend Snippet: Fig. 1. The concept of using CD146 target gold nanorods (AuNRs) combined with mild hyperthermia to disrupt the actin cytoskeleton and migration of the cancer cells. On the surfaces of metastatic melanoma and breast cancer cells, transmembrane CD146 not only serves as an ‘‘anchor” to recruit actin cytoskeleton to cell membrane through ERM proteins (i.e., the ‘‘chain”) to modulate the cancer cell migration, but also activates the highly conserved ERM proteins in cytosol by phosphorylation, allowing activated ERM (phosphorylated ERM, p-ERM) proteins to translocate from the cytosol to the membrane-cytoskeleton interface. Two hypotheses will be tested in this work: (i) The uptake of CD146 targeted gold nanorods will deplete the cell membrane CD146, and thereby disrupt the actin cytoskeleton and cell migration (i.e., remove the ‘‘anchor”); and (ii) Mild hyperthermia will interfere with the phosphorylation of ERM proteins to further impair migration (i.e., break the ‘‘chain”).

Article Snippet: AlexaFluor594 conjugated human MCAM/CD146 antibody and biotinylated human MCAM/ CD146 antibody were purchased from R&D system.

Techniques: Migration, Membrane, Phospho-proteomics

Journal: Journal of colloid and interface science

Article Title: Targeting cancer cell adhesion molecule, CD146, with low-dose gold nanorods and mild hyperthermia disrupts actin cytoskeleton and cancer cell migration.

doi: 10.1016/j.jcis.2021.05.144

Figure Lengend Snippet: Fig. 2. Preparation and characterization of CD146 targeted gold nanorods. (A) Schematic illustrating the surface functionalization of gold nanorods (AuNRs) with CD146 antibody. (B) TEM images of CTAB-capped AuNRs. (C) Extinction spectra of AuNRs following surface functionalization steps. (D) Zeta potential of AuNRs following surface functionalization steps. Error bars are standard deviation of three independent experiments (n = 3). (E) TEM images of CD146 antibody functionalized AuNRs. A clear coating can be observed surrounding the AuNR surfaces. (F) Quantitative analysis of length and diameter of AuNRs before and after the surface functionalization, confirming the successful surface functionalization of AuNRs.

Article Snippet: AlexaFluor594 conjugated human MCAM/CD146 antibody and biotinylated human MCAM/ CD146 antibody were purchased from R&D system.

Techniques: Zeta Potential Analyzer, Standard Deviation

Journal: Journal of colloid and interface science

Article Title: Targeting cancer cell adhesion molecule, CD146, with low-dose gold nanorods and mild hyperthermia disrupts actin cytoskeleton and cancer cell migration.

doi: 10.1016/j.jcis.2021.05.144

Figure Lengend Snippet: Fig. 7. Mechanistic investigation of the effect of CD-146 target gold nanorods with mild hyperthermia on the actin cytoskeleton structures of cancer cells. (A) and (B) Western blot analysis of membrane CD146 on both cell lines with and without treatments. Sodium potassium ATPase is used as a membrane housekeeping protein. (C) and (D) Western blot analysis of total ERM and p-ERM levels in both cell lines with and without treatments. b-actin is used as a house-keeping protein in cytosol. Data is mean ± standard deviation, n = 3. Student’s t-test, *P < 0.05, ***P < 0.001.

Article Snippet: AlexaFluor594 conjugated human MCAM/CD146 antibody and biotinylated human MCAM/ CD146 antibody were purchased from R&D system.

Techniques: Western Blot, Membrane, Standard Deviation

Journal: American Journal of Respiratory and Critical Care Medicine

Article Title: Characterization of GDF2 Mutations and Levels of BMP9 and BMP10 in Pulmonary Arterial Hypertension

doi: 10.1164/rccm.201906-1141oc

Figure Lengend Snippet: Figure 1: Characterisation of Expressed BMP9 Mutant Proteins. (a) Schematic of

Article Snippet: After washing, 0.04 μg/well of anti-human BMP9 detection antibody (R&D Systems BAF3209) was added in Copyright © 2019 by the American Thoracic Society 6 PBS/1% BSA containing 0.2% GS.

Techniques: Mutagenesis

Journal: American Journal of Respiratory and Critical Care Medicine

Article Title: Characterization of GDF2 Mutations and Levels of BMP9 and BMP10 in Pulmonary Arterial Hypertension

doi: 10.1164/rccm.201906-1141oc

Figure Lengend Snippet: Figure 2: Loss of Activity in Pro:BMP9 Mutants Predicted to be Pathogenic. (a,b)

Article Snippet: After washing, 0.04 μg/well of anti-human BMP9 detection antibody (R&D Systems BAF3209) was added in Copyright © 2019 by the American Thoracic Society 6 PBS/1% BSA containing 0.2% GS.

Techniques: Activity Assay

Journal: American Journal of Respiratory and Critical Care Medicine

Article Title: Characterization of GDF2 Mutations and Levels of BMP9 and BMP10 in Pulmonary Arterial Hypertension

doi: 10.1164/rccm.201906-1141oc

Figure Lengend Snippet: Figure 3: Loss of active BMP9 in PAH patients carrying putatively pathogenic GDF2

Article Snippet: After washing, 0.04 μg/well of anti-human BMP9 detection antibody (R&D Systems BAF3209) was added in Copyright © 2019 by the American Thoracic Society 6 PBS/1% BSA containing 0.2% GS.

Techniques:

Journal: American Journal of Respiratory and Critical Care Medicine

Article Title: Characterization of GDF2 Mutations and Levels of BMP9 and BMP10 in Pulmonary Arterial Hypertension

doi: 10.1164/rccm.201906-1141oc

Figure Lengend Snippet: Figure 4: Plasma BMP9 and pBMP10 levels are not reduced in PAH, but a subset of

Article Snippet: After washing, 0.04 μg/well of anti-human BMP9 detection antibody (R&D Systems BAF3209) was added in Copyright © 2019 by the American Thoracic Society 6 PBS/1% BSA containing 0.2% GS.

Techniques: Clinical Proteomics

Journal: The Journal of investigative dermatology

Article Title: Activin A is anti-lymphangiogenic in a melanoma mouse model.

doi: 10.1038/jid.2014.328

Figure Lengend Snippet: Figure 3. Activin does not interfere with melanoma metastasis. (a) Examples for anti-Lyve-1 stainings (red color) for lymphatic vessels in primary melanoma overexpressing indicated genes; arrows indicate lymphatic vessels filled with tumor cells in control and INHBA tumors, but narrow and empty vessels in Wnt1 þ

Article Snippet: Goat anti-mouse Wnt1, rabbit anti-human INHBA, and goat anti- human VEGF-C antibodies were from R&D Systems (Minneapolis, MN); mouse anti-human FST was from Abcam (Cambridge, UK); rabbit anti-human Phospho-Smad2 was from Cell Signal (Cell Signaling Technology, Danvers, MA); rabbit anti-mouse Lyve-1 was from Acris (Herford, Germany); rabbit anti-mosue CD31 was from Neomarkers (Fremont, CA); mouse anti-human vimentin (clone V9) was from Dako (Glostrup, Denmark); biotinylated secondary anti- bodies were from Vector Laboratories (Burlingame, CA); horseradish peroxidase rabbit anti-goat IgG was from Zymax (Novex, Life Technologies, Carlsbad, CA); human recombinant TGFb was from PeproTech (Rocky Hill, NJ); and human recombinant Wnt3a and (activin A) ActA were from R&D Systems.

Techniques: Control