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mouse anti umod biotinylated antibody 774 (R&D Systems)

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R&D Systems mouse anti umod biotinylated antibody 774
Mouse Anti Umod Biotinylated Antibody 774, supplied by R&D Systems, used in various techniques. Bioz Stars score: 93/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/product/mouse+anti+umod+biotinylated+antibody/10__1172_slash_jci183343-327-12-18?v=R%26D+Systems
Average 93 stars, based on 4 article reviews

mouse anti umod biotinylated antibody 774 - by Bioz Stars, 2026-07

93/100 stars

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( 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.

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

( 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.

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

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.

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

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

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

( 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.

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

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.

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

( 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.

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

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.

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

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

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

Figure 3. The tubular injury marker KIM-1 is higher in mice lacking in UMOD during obstructive nephropathy. By western blotting, kidney KIM-1 protein levels were increased after UUO (n = 5 for sham groups; n = 7 for UUO groups) at all time-points (days 7, 14, and 21); the levels were signiﬁcantly higher in the UMOD/ groups. Results are corrected for protein loading using GAPDH levels. (A, B). Kidney KIM-1 mRNA levels measured by real-time PCR (n = 3 for sham groups; n = 5 for UUO groups) were signiﬁcantly higher on UUO days 14 and 21 (C). Representative day 14 immunoﬂuorescence photomicrographs illustrate more intense KIM-1 staining in the UMOD/ kidney (D). KIM-1 staining was concentrated along the apical membrane with lesser amounts in tubular lumina; the latter more evident in the UMOD+/+ mice. Brightly stained ribbon-like KIM-1 + bands, frequently observed along the apical membrane in the UMOD-/- mice, were much less common in the UMOD+/+ mice. Dual staining for UMOD (green) and KIM-1 (red) in a UMOD+/+ day 14 kidney illustrates an area of tubular intraluminal staining for both proteins (yellow; highlighted by the arrow) (E). Dual staining for UMOD (green) and the proximal tubular apical membrane protein LRP2 (red) in a UMOD+/+ day 14 kidney illustrates the presence of luminal UMOD deposits surrounded by LRP2 + tubules (arrows) (F). Bar graphs represent means +1SEM; * indicates a P value < 0.05. Photomicrograph magniﬁcation: 940. Scale bars: 25 lm.

Journal: Physiological reports

Article Title: Uromodulin deficiency alters tubular injury and interstitial inflammation but not fibrosis in experimental obstructive nephropathy.

doi: 10.14814/phy2.13654

Figure Lengend Snippet: Figure 3. The tubular injury marker KIM-1 is higher in mice lacking in UMOD during obstructive nephropathy. By western blotting, kidney KIM-1 protein levels were increased after UUO (n = 5 for sham groups; n = 7 for UUO groups) at all time-points (days 7, 14, and 21); the levels were signiﬁcantly higher in the UMOD/ groups. Results are corrected for protein loading using GAPDH levels. (A, B). Kidney KIM-1 mRNA levels measured by real-time PCR (n = 3 for sham groups; n = 5 for UUO groups) were signiﬁcantly higher on UUO days 14 and 21 (C). Representative day 14 immunoﬂuorescence photomicrographs illustrate more intense KIM-1 staining in the UMOD/ kidney (D). KIM-1 staining was concentrated along the apical membrane with lesser amounts in tubular lumina; the latter more evident in the UMOD+/+ mice. Brightly stained ribbon-like KIM-1 + bands, frequently observed along the apical membrane in the UMOD-/- mice, were much less common in the UMOD+/+ mice. Dual staining for UMOD (green) and KIM-1 (red) in a UMOD+/+ day 14 kidney illustrates an area of tubular intraluminal staining for both proteins (yellow; highlighted by the arrow) (E). Dual staining for UMOD (green) and the proximal tubular apical membrane protein LRP2 (red) in a UMOD+/+ day 14 kidney illustrates the presence of luminal UMOD deposits surrounded by LRP2 + tubules (arrows) (F). Bar graphs represent means +1SEM; * indicates a P value < 0.05. Photomicrograph magniﬁcation: 940. Scale bars: 25 lm.

Article Snippet: Primary antibodies used in the study were: sheep-anti mouse UMOD, goat-anti-mouse KIM-1, mouse-antihuman KIM-1, goat-anti mouse NGAL (R&D Systems Inc, Minneapolis, MN, USA); sheep-anti-human UMOD, rat anti-mouse F4/80 (AbD Serotec/Bio-Rad, Mississauga, ON, Canada); mouse anti-mouse aSMA, rabbit antihuman fibronectin (Sigma-Aldrich, St. Louis, MO, USA); rabbit anti-mouse/human/rat TRPV5 (Alomone Labs, Jerusalem, Israel); mouse anti-human PCNA (eBioscience/ Thermo Fisher Scientific, Waltham, MA, USA); rabbit anti-mouse/human/monkey megalin/LRP-2 (Abcam, Toronto, ON, Canada); rabbit-anti-mouse Laminin (Millipore/Sigma), mouse-anti-mammalian acetylated a-Tubulin (Santa Cruz Biotechnology, Dallas, TX, USA), and rabbit anti-mouse GAPDH (Cell Signaling Technology, Danvers, MA, USA).

Techniques: Marker, Western Blot, Real-time Polymerase Chain Reaction, Staining, Membrane

Figure 4. Effect of UMOD on other tubular injury markers in UUO nephropathy. Kidney NGAL protein levels, measured by western blotting, were elevated after UUO (n = 5 for sham groups; n = 7 for UUO groups) at all time-points (days 7, 14, and 21); differences between the genotypes were only found on day 21, when they were signiﬁcantly higher in the UMOD+/+ group (A, B). Kidney TRPV5 mRNA levels are markedly upregulated after UUO (n = 3 for sham groups; n = 5 for UUO groups); at all time-points (days 7, 14, and 21) mRNA levels were signiﬁcantly higher in the UMOD+/+ groups (C). By dual staining ﬂuorescence microscopy, TRPV5 (red) was almost exclusively found in tubules with intraluminal UMOD deposits (green), as shown for a day 14 UUO UMOD+/+ kidney (D). Bar graphs represent means +1SEM; * indicates P value < 0.05. Photomicrograph magniﬁcation: 940. Scale bar: 25 lm.

Journal: Physiological reports

Article Title: Uromodulin deficiency alters tubular injury and interstitial inflammation but not fibrosis in experimental obstructive nephropathy.

doi: 10.14814/phy2.13654

Figure Lengend Snippet: Figure 4. Effect of UMOD on other tubular injury markers in UUO nephropathy. Kidney NGAL protein levels, measured by western blotting, were elevated after UUO (n = 5 for sham groups; n = 7 for UUO groups) at all time-points (days 7, 14, and 21); differences between the genotypes were only found on day 21, when they were signiﬁcantly higher in the UMOD+/+ group (A, B). Kidney TRPV5 mRNA levels are markedly upregulated after UUO (n = 3 for sham groups; n = 5 for UUO groups); at all time-points (days 7, 14, and 21) mRNA levels were signiﬁcantly higher in the UMOD+/+ groups (C). By dual staining ﬂuorescence microscopy, TRPV5 (red) was almost exclusively found in tubules with intraluminal UMOD deposits (green), as shown for a day 14 UUO UMOD+/+ kidney (D). Bar graphs represent means +1SEM; * indicates P value < 0.05. Photomicrograph magniﬁcation: 940. Scale bar: 25 lm.

Techniques: Western Blot, Staining, Microscopy

Figure 9. UMOD reduces tubular cell KIM-1 levels in vitro. Preliminary studies to maximize the exposure of KIM-1 producing 769-P cells to UMOD demonstrate that both 769-P and 293 cells transiently transfected with a pcDNA3.1 vector expressing human UMOD (pcDNA3.1-UMOD) produced readily detectable UMOD protein levels within cells and their conditioned medium compared with cells transfected with a pcDNA3.1 vector expressing a noncoding control vector (pcDNA3.1-C) (A). Under transwell culture conditions, 769-P cells transfected with pcDNA3.1-UMOD (769-P- UMOD) and harvested from the upper chamber after exposure to UMOD from both the upper and lower chambers for 48 h, expressed signiﬁcantly lower KIM-1 protein levels compared with cells transfected with pcDNA3.1-C (769-P-C) and not exposed to UMOD (B). Lower chambers contained 293 cells transfected with pcDNA3.1-C (293-C) or pcDNA3.1-UMOD (293-UMOD). Results are western blot data corrected for GAPDH protein levels (n = 3). KIM- 1 mRNA levels in 769-P cells cultured under the same conditions (n = 3) were similar between control transfected and UMOD transfected cells (C). * indicates P value < 0.05.

Journal: Physiological reports

Article Title: Uromodulin deficiency alters tubular injury and interstitial inflammation but not fibrosis in experimental obstructive nephropathy.

doi: 10.14814/phy2.13654

Figure Lengend Snippet: Figure 9. UMOD reduces tubular cell KIM-1 levels in vitro. Preliminary studies to maximize the exposure of KIM-1 producing 769-P cells to UMOD demonstrate that both 769-P and 293 cells transiently transfected with a pcDNA3.1 vector expressing human UMOD (pcDNA3.1-UMOD) produced readily detectable UMOD protein levels within cells and their conditioned medium compared with cells transfected with a pcDNA3.1 vector expressing a noncoding control vector (pcDNA3.1-C) (A). Under transwell culture conditions, 769-P cells transfected with pcDNA3.1-UMOD (769-P- UMOD) and harvested from the upper chamber after exposure to UMOD from both the upper and lower chambers for 48 h, expressed signiﬁcantly lower KIM-1 protein levels compared with cells transfected with pcDNA3.1-C (769-P-C) and not exposed to UMOD (B). Lower chambers contained 293 cells transfected with pcDNA3.1-C (293-C) or pcDNA3.1-UMOD (293-UMOD). Results are western blot data corrected for GAPDH protein levels (n = 3). KIM- 1 mRNA levels in 769-P cells cultured under the same conditions (n = 3) were similar between control transfected and UMOD transfected cells (C). * indicates P value < 0.05.

Techniques: In Vitro, Transfection, Plasmid Preparation, Expressing, Produced, Control, Western Blot, Cell Culture

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