rabbit polyclonal anti nmnat1  (Proteintech)

Journal: International Journal of Molecular Sciences

Article Title: Nmnat1 Deficiency Causes Mitoribosome Excess in Diabetic Nephropathy Mediated by Transcriptional Repressor HIC1

doi: 10.3390/ijms25126384

Figure Lengend Snippet: Reduced NMNAT1 levels in db/db mice and human renal fibrosis of DN. ( a ) Immunolocalization of NMNAT1 in the kidneys of nondiabetic control mice (db/m) and diabetic mice (db/db) at 32 weeks of age. The protein expression in the control and diabetic kidneys was examined through immunohistochemistry; representative images are shown. Positive protein expression was stained brown by a 3,3′-diaminobenzidine agent. Scale bar, 50 µm; N = 7. Results of immunohistochemical scoring assessing whole kidneys are expressed as the mean ± standard error of the mean (SEM). * p < 0.05. In addition, we have scored the Nmnat1 levels in tubular and glomerular areas separately. All data are depicted as the mean ± SEM. Horizontal bars denote statistically significant differences between the two groups. * p < 0.05. ( b ) Real-time quantitative reverse transcription–polymerase chain reaction (RT–PCR) analysis of the renal mRNA of NMNAT1, NMNAT2, and NMNAT3. The kidney tissue specimens for RT–PCR were derived from db/m and db/db mice at 32 weeks of age. N = 7 mice per group. Glyceraldehyde 3-phosphate dehydrogenase was used as a control. All data are depicted as the mean ± SEM. Horizontal bars denote statistically significant differences between the two groups. * p < 0.05. ( c ) Masson’s trichrome and NMNAT1 immunostaining in human kidneys. Representative photomicrographs of needle renal biopsy specimens from patients with DN with mild or severe fibrosis. The relationship between the Masson’s trichrome stain-positive area and the immunostaining intensity for NMNAT1 in renal biopsy specimens from patients with DN (N = 11). Pearson’s correlation analysis was used to calculate r and p values. Bars; 50 nm.

Article Snippet: We conducted dual labeling by incubating overnight 5-μm thick cryostat kidney sections mixed with two primary antibodies, namely, rabbit polyclonal anti- Nmnat1 (1:50, 11399-1AP, Proteintech, Chicago, IL, USA) and mouse polyclonal anti-aquaporin-1 (1:100, B-11, Santa Cruz Biotechnology, Santa Cruz, CA, USA).

Techniques: Control, Expressing, Immunohistochemistry, Staining, Immunohistochemical staining, Reverse Transcription, Polymerase Chain Reaction, Reverse Transcription Polymerase Chain Reaction, Derivative Assay, Immunostaining

Journal: International Journal of Molecular Sciences

Article Title: Nmnat1 Deficiency Causes Mitoribosome Excess in Diabetic Nephropathy Mediated by Transcriptional Repressor HIC1

doi: 10.3390/ijms25126384

Figure Lengend Snippet: Clinical data of patients with diabetic nephropathy. eGFR, estimated glomerular filtration rate; HbA1c, glycosylated hemoglobin.

Article Snippet: We conducted dual labeling by incubating overnight 5-μm thick cryostat kidney sections mixed with two primary antibodies, namely, rabbit polyclonal anti- Nmnat1 (1:50, 11399-1AP, Proteintech, Chicago, IL, USA) and mouse polyclonal anti-aquaporin-1 (1:100, B-11, Santa Cruz Biotechnology, Santa Cruz, CA, USA).

Techniques: Filtration

Journal: International Journal of Molecular Sciences

Article Title: Nmnat1 Deficiency Causes Mitoribosome Excess in Diabetic Nephropathy Mediated by Transcriptional Repressor HIC1

doi: 10.3390/ijms25126384

Figure Lengend Snippet: Effects of Nmnat1 CKO on urinary albuminuria. ( a ) Nmnat1 immunofluorescence intensity levels in each mouse group. Kidney tissue specimens obtained from each 32-week-old mouse were stained using immunofluorescence for Nmnat1 (green) and AQP1 (red). ( b ) The panel shows the results of the quantitative analysis of Nmnat1 fluorescence intensity. Scale bar, 50 µm; N = 7. ( c ) Real-time quantitative reverse transcription–PCR analysis of renal mRNA of Nmnat1 (N = 7) in control and CKO mice at 32 weeks of age. ( d ) Temporal changes in the mean plasma glucose concentrations in mice from each group. We assessed the mice at 8, 16, 24, and 32 weeks of age. N = 7 mice per group. ( e ) Changes in mouse body weight from 8 to 32 weeks of age. N = 7 mice per group. Horizontal bars indicate statistically significant differences in each group. * p < 0.05. ( f ) Serum creatine levels in each mouse at 32 weeks of age. N = 6 mice per group. ( g ) Urinary albumin excretion in each mouse at 32 weeks of age. N = 6 mice per group. ( h ) Sodium dodecyl-sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) of mouse urine samples. Urine samples of mice from each group at 32 weeks of age was tested using 15% SDS–PAGE before staining with Coomassie blue. N = 2 mice per group. AQP1, anti-aquaporin-1; Nmnat1 , nicotinamide mononucleotide adenylyl transferase1; PCR, polymerase chain reaction; Cont, control; CKO, conditional knockout. ( i ) Representative histological findings of the kidneys of control and CKO mice via Masson’s trichrome staining. Scale bar = 500 nm. Percentage of renal fibrosis area (N = 20 mice per group). Horizontal bars indicate statistically significant differences in each group. * p < 0.05. ( j ) Renal tissue concentrations of NAD + metabolites, NAM, NMN, and NAD at 32 weeks of age in the control and CKO groups (N = 7). Statistical significance between each group is represented by a horizontal bar. * p < 0.05.

Article Snippet: We conducted dual labeling by incubating overnight 5-μm thick cryostat kidney sections mixed with two primary antibodies, namely, rabbit polyclonal anti- Nmnat1 (1:50, 11399-1AP, Proteintech, Chicago, IL, USA) and mouse polyclonal anti-aquaporin-1 (1:100, B-11, Santa Cruz Biotechnology, Santa Cruz, CA, USA).

Techniques: Immunofluorescence, Staining, Fluorescence, Reverse Transcription, Control, Clinical Proteomics, Polyacrylamide Gel Electrophoresis, SDS Page, Polymerase Chain Reaction, Knock-Out

Journal: International Journal of Molecular Sciences

Article Title: Nmnat1 Deficiency Causes Mitoribosome Excess in Diabetic Nephropathy Mediated by Transcriptional Repressor HIC1

doi: 10.3390/ijms25126384

Figure Lengend Snippet: Renal phenotypes in Nmnat1 CKO mice. ( a ) Representative images of albumin staining in each mouse. Arrows denote albumin cast in CKO mice. The right panel shows the relative staining intensity. N = 7 mice per group. ( b ) Changes in albumin uptake-related molecules in Pck1 CKO mice. The kidney tissue specimens for RT–PCR were derived from CKO and control mice at 32 weeks of age. N = 7 mice per group. Real-time quantitative reverse transcription–polymerase chain reaction (RT–PCR) analysis of renal mRNA of megalin, cubilin, and amnionless. Glyceraldehyde 3-phosphate dehydrogenase was used as a control. All data are depicted as the mean ± standard error of the mean. Horizontal bars denote statistically significant differences between the two groups. * p < 0.05. ( c ) Representative immunofluorescence double staining of TUNEL (green) and AQP1 (red, proximal tubules) in kidney tissues (N = 7). Scale bars, 50 µm. The kidney tissue specimens were derived from Cont and CKO mice at 32 weeks of age. White arrows denote apoptotic tubular cells.

Article Snippet: We conducted dual labeling by incubating overnight 5-μm thick cryostat kidney sections mixed with two primary antibodies, namely, rabbit polyclonal anti- Nmnat1 (1:50, 11399-1AP, Proteintech, Chicago, IL, USA) and mouse polyclonal anti-aquaporin-1 (1:100, B-11, Santa Cruz Biotechnology, Santa Cruz, CA, USA).

Techniques: Staining, Reverse Transcription Polymerase Chain Reaction, Derivative Assay, Control, Reverse Transcription, Polymerase Chain Reaction, Immunofluorescence, Double Staining, TUNEL Assay

Journal: International Journal of Molecular Sciences

Article Title: Nmnat1 Deficiency Causes Mitoribosome Excess in Diabetic Nephropathy Mediated by Transcriptional Repressor HIC1

doi: 10.3390/ijms25126384

Figure Lengend Snippet: Mitoribosome excess in Nmnat1 CKO mice. ( a ) Representative photomicrographs showing TGF-β immunostaining in each group. The bar graph represents the quantitative analysis of TGF-β staining. N = 7 mice per group. Scale bar = 100 μm. ( b ) Representative photomicrographs indicate collagen IV immunostaining in each group. The bar graph shows the quantitative analysis of collagen IV-stained areas. N = 7 mice per group. Scale bar = 100 μm. Kidney tissue specimens for immunostaining were derived from the four 32-week-old mouse groups. All data are depicted as mean ± standard error of the mean. Horizontal bars indicate statistically significant differences in each group. * p < 0.05. ( c ) Representative electron micrograph in each group. Scale bar = 500 nm. Blue squares indicate the enlarged regions. Expanded images are also presented. Blue arrowheads indicate mitoribosomes. Scale bar = 500 nm. Illustration depicts mitoribosome excess in CKO mice. The number of mitoribosomes was intact in Cont mice. For electron microscopy, the kidney tissue specimens were embedded into Epon epoxy resin. Electron micrographs of 10 proximal tubules (PTs) per kidney were randomly obtained for each mouse to evaluate the morphometry of PTs. The red symbols represent mitoribosomes. ( d ) Real-time quantitative reverse transcription–polymerase chain reaction (RT–PCR) analysis of the renal mRNA of CRIF1, a mitoribosomal synthesis regulator. Glyceraldehyde 3-phosphate dehydrogenase was used as a control. The kidney tissue specimens for RT–PCR were obtained from CKO and control mice at 32 weeks of age. N = 7 mice per group. ( e ) Real-time quantitative reverse transcription–polymerase chain reaction (RT–PCR) analysis of the renal mRNA of intracellular organelle markers. MRPL13 and MRPS15 are mitoribosomal proteins, VDAC is a mitochondrial protein, and Lamin B is a nuclear protein. Glyceraldehyde 3-phosphate dehydrogenase was used as a control. For RT–PCR, the kidney tissue specimens were obtained from CKO and control mice at 32 weeks of age. N = 7 mice per group.

Article Snippet: We conducted dual labeling by incubating overnight 5-μm thick cryostat kidney sections mixed with two primary antibodies, namely, rabbit polyclonal anti- Nmnat1 (1:50, 11399-1AP, Proteintech, Chicago, IL, USA) and mouse polyclonal anti-aquaporin-1 (1:100, B-11, Santa Cruz Biotechnology, Santa Cruz, CA, USA).

Techniques: Immunostaining, Staining, Derivative Assay, Electron Microscopy, Reverse Transcription, Polymerase Chain Reaction, Reverse Transcription Polymerase Chain Reaction, Control

Journal: International Journal of Molecular Sciences

Article Title: Nmnat1 Deficiency Causes Mitoribosome Excess in Diabetic Nephropathy Mediated by Transcriptional Repressor HIC1

doi: 10.3390/ijms25126384

Figure Lengend Snippet: CRIF1 gene regulation by HIC1 and SP1. ( a ) Localization and nucleotide sequence in the murine CRIF1 promoter region. The red characters represent the effects of Nmnat1 deficiency–mediated by two putative HIC1-responsive element (HiREs) on CRIF1 upregulation. The transcription start sites are indicated in gray. ( b ) The schematic diagram describes seven deletion mutants in the CRIF1 promoter sequences (−1955, −1263, −813, −649, −496, −128, and −15) that were cloned upstream from a luciferase reporter gene. The bar graphs present the results of transient transfection of cultured proximal tubules (PTs), illustrating the promoter activities associated with each deletion. Luciferase activity is shown relative to that of the −1955 Luc vector in the control vector-transfected cells. Values are expressed as the mean ± standard error of the mean (SEM). * p < 0.05 vs. each Luc transfected PTs (N = 3 independent experiments). ( c ) Mutation analysis of CRIF1 promoter activity in cultured PT cells. −649 Luc, WT Nmnat1 promoter; M1, proximal HiRE mutation; M2, distal HiRE mutation; M3, mutation in both HiREs corresponding to the HIC1 binding sites. * p < 0.05 vs. −649 Luc in control cells (N = 3 independent experiments). ( d ) Representative photomicrographs showing HIC1 immunostaining in each group. The bar graph illustrates the results of quantitative analysis. N = 7 mice per group. Light micrograph; scale bar = 100 μm. Kidney tissue specimens for immunostaining were obtained from four mouse groups at 32 weeks of age. All data are shown as mean ± SEM. Horizontal bars indicate statistically significant differences in each group. * p < 0.05. ( e ) Schematic representation of the murine CRIF1 gene and promoter. The solid boxes indicate the binding of HiRE to HIC1 and binding of GC box to Sp1, highlighted in red and yellow, respectively. Cont, control; CKO, conditional knockout; CRIF1, CR6-interacting factor 1; Dmrt1, double sex and mab-3 related transcription factor 1; Egr2, early growth response protein 2; Nr5A2, nuclear receptor subfamily 5 group A member 2; HIC1, HIC ZBTB transcriptional repressor 1; STAT2, signal transducer and activator of transcription 2; Sp1, specificity protein 1; Luc, luciferase; Nmnat , nicotinamide mononucleotide adenylyl transferase; DN, diabetic nephropathy.

Article Snippet: We conducted dual labeling by incubating overnight 5-μm thick cryostat kidney sections mixed with two primary antibodies, namely, rabbit polyclonal anti- Nmnat1 (1:50, 11399-1AP, Proteintech, Chicago, IL, USA) and mouse polyclonal anti-aquaporin-1 (1:100, B-11, Santa Cruz Biotechnology, Santa Cruz, CA, USA).

Techniques: Sequencing, Clone Assay, Luciferase, Transfection, Cell Culture, Activity Assay, Plasmid Preparation, Control, Mutagenesis, Binding Assay, Immunostaining, Knock-Out

Journal: International Journal of Molecular Sciences

Article Title: Nmnat1 Deficiency Causes Mitoribosome Excess in Diabetic Nephropathy Mediated by Transcriptional Repressor HIC1

doi: 10.3390/ijms25126384

Figure Lengend Snippet: Mitoribosomal dysfunction and mitochondrial dysfunction in CKO mice. ( a ) Western blot analysis of renal protein levels of OXPHOS subunits encoded by nDNA and mtDNA. The kidney tissue specimens were obtained from CKO and control mice at 32 weeks of age. The results of a representative experiment among the three performed are shown. All data are indicated as the mean ± standard error of the mean (SEM). Horizontal bars denote statistically significant differences between the two groups. * p < 0.05. The bar graph in the lower panels illustrates the quantification of the band intensity. Glyceraldehyde 3-phosphate dehydrogenase was used as a control. N = 3 mice per group. ( b ) Real-time quantitative RT–PCR analysis of the renal mRNA of mitoribosomal translational regulators. Glyceraldehyde 3-phosphate dehydrogenase was used as a control. For RT–PCR, the kidney tissue specimens were obtained from CKO and control mice at 32 weeks of age. N = 7 mice per group. ( c ) Illustration depicting the dysfunctional mitoribosomes and their concomitant mitochondrial dysfunction in CKO mice. These functions are intact in control mice. ( d ) The oxygen consumption rate (OCR) of TECs isolated from CKO and control mice was measured using a Seahorse XF-24 flux analyzer. N = 3. ( e ) The ratio of red/green fluorescence of JC-1 in TECs isolated from CKO and control mice was used as a measure of mitochondrial membrane potential. N = 7. ( f ) Fluorescence of MitoSox in TECs isolated from CKO and control mice as a measure of mitochondrial levels of reactive oxygen species. N = 7. ( g ) ATP content in TECs isolated from CKO and control mice. N = 7. All data are presented as mean ± SEM. Horizontal bars indicate statistically significant differences between the two groups. * p < 0.05. ( h ) Scheme depicting the new mitoribosome excess-mediated mechanism of renal profibrotic changes and mitochondrial dysfunction in CKO mice. The downregulation of Nmnat1 expression decreased HIC1 expression, resulting in increased CRIF1 expression and ultimately mitoribosome excess. The upregulation of mitoribosome excess leads to mitoribosomal dysfunction, deposition of collagen IV in addition to OXPHOS impairment and tubular mitochondrial dysfunction. MRPL13, mitochondrial ribosomal protein L13; MRPS15, mitochondrial ribosomal protein S15; VDAC, voltage-dependent anion channel; Cont, control; CKO, conditional knockout; OXPHOS, oxidative phosphorylation; ND1, NADH-ubiquinone oxidoreductase chain 1; NDUFA9, NADH: ubiquinone oxidoreductase subunit A9; FP, fluorescent protein; Cyto b, cytochrome b; UQCRC2, ubiquinol-cytochrome c reductase core protein 2; COX1, cytochrome c oxidase 1; ATP8, adenosine triphosphate 8; ATP5A1, ATP synthase F1 subunit alpha; mtIF3, mitochondrial translational initiation factor 3; mtEFTu, mitochondrial elongation factor EFTu; mtRRF, mitochondrial ribosome recycling factor; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; CCCP, carbonyl cyanide m-chlorophenyl hydrazone.

Article Snippet: We conducted dual labeling by incubating overnight 5-μm thick cryostat kidney sections mixed with two primary antibodies, namely, rabbit polyclonal anti- Nmnat1 (1:50, 11399-1AP, Proteintech, Chicago, IL, USA) and mouse polyclonal anti-aquaporin-1 (1:100, B-11, Santa Cruz Biotechnology, Santa Cruz, CA, USA).

Techniques: Western Blot, Control, Quantitative RT-PCR, Reverse Transcription Polymerase Chain Reaction, Isolation, Fluorescence, Membrane, Expressing, Knock-Out, Phospho-proteomics