polyclonal sheep anti human ssbp1 antibodies  (R&D Systems)

Journal: The Journal of Clinical Investigation

Article Title: Dominant mutations in mtDNA maintenance gene SSBP1 cause optic atrophy and foveopathy

doi: 10.1172/JCI128513

Figure Lengend Snippet: ( A ) Pedigree showing males (squares) and females (circles) of the families carrying the pathogenic c.113G>A variant (families A and B) or c.320G>A (families C, D, and E) in the SSBP1 gene. Black symbols denote affected family members, and white symbols denote unaffected family members. The mutation status of each analyzed family member is indicated. ( B ) Electropherograms of genomic DNA sequencing from family A (left panel) and family D (right panel). WT and mutant (Mut) alleles are indicated. All patients were heterozygous for the identified mutation. ( C ) Sequence alignments showing conservation of the 2 affected amino acid residues between different species. ( D ) Schematic representation of the human SSBP1 (gene on the top, protein on the bottom) with the localization of the 2 mutations. Red squares represent exons, and regions corresponding to the mitochondrial transit domain (blue) and DNA-binding domain (green) are shown.

Article Snippet: Membranes were saturated with 5% nonfat milk dissolved in 0.1% Tween-TBS for 2 hours at room temperature, then incubated overnight at 4°C with polyclonal sheep anti-SSBP1 (1 μg/ml, catalog AF6588, Bio-techne), monoclonal mouse anti-Grim19 (1:1000, catalog ab110240, Abcam), anti-SDHA (1:1000, catalog ab14715, Abcam), anti-UQCRCII (1:1000, catalog ab14745, Abcam), anti-MTCO1 (1:1000, catalog ab14705, Abcam), anti-GAPDH (1:2000, catalog G8795, MilliporeSigma), and polyclonal rabbit anti-ATP synthase (1:1000, catalog ab151229, Abcam).

Techniques: Variant Assay, Mutagenesis, DNA Sequencing, Sequencing, Binding Assay

Journal: The Journal of Clinical Investigation

Article Title: Dominant mutations in mtDNA maintenance gene SSBP1 cause optic atrophy and foveopathy

doi: 10.1172/JCI128513

Figure Lengend Snippet: ( A ) SSBP1 protein expression in human retina. Immunofluorescence labeling was done on retinal cross-sections from healthy human donor. Hoechst was added to label nuclei (blue). SSBP1 localization was done using a specific antibody (green). An antibody against the ATP synthase subunit 5A was used to target mitochondria (red). RPE, retinal pigment epithelium; POS, photoreceptor outer segment; ONL, outer nuclear layer; INL, inner nuclear layer; GCL, ganglion cell layer. Scale bars: 20 μm. ( B ) Quantification of SSBP1 transcript levels in cultured skin fibroblasts from both controls and affected individuals (patient 1, patient 2, and patient 3). mRNA levels were normalized to the reference gene L27 . ( C ) Western blot in lysates from controls (control 1 and control 2) and patient fibroblasts and densitometric analysis of SSBP1 protein abundance in lysates from both controls and patient fibroblasts. GAPDH was used as a loading control. Data are shown as mean ± SEM. *** P < 0.001. ( D ) Representative ultrastructure of the mitochondria from both controls (C1, C2) and patient fibroblasts by TEM. Scale bar: 1 μm. Single arrows show abnormal mitochondria. Double arrows show lipid droplets. Quantification analyses of abnormal mitochondria (large vacuoles, disturbed cristae) in fibroblasts from both controls and patients. Data are represented as mean percentage of abnormal mitochondria ± SEM in total examined mitochondria. ** P < 0.01. All data are representative of 3 independent experiments. One-way ANOVA with Dunnett’s correction was used.

Article Snippet: Membranes were saturated with 5% nonfat milk dissolved in 0.1% Tween-TBS for 2 hours at room temperature, then incubated overnight at 4°C with polyclonal sheep anti-SSBP1 (1 μg/ml, catalog AF6588, Bio-techne), monoclonal mouse anti-Grim19 (1:1000, catalog ab110240, Abcam), anti-SDHA (1:1000, catalog ab14715, Abcam), anti-UQCRCII (1:1000, catalog ab14745, Abcam), anti-MTCO1 (1:1000, catalog ab14705, Abcam), anti-GAPDH (1:2000, catalog G8795, MilliporeSigma), and polyclonal rabbit anti-ATP synthase (1:1000, catalog ab151229, Abcam).

Techniques: Expressing, Immunofluorescence, Labeling, Cell Culture, Western Blot

Journal: The Journal of Clinical Investigation

Article Title: Dominant mutations in mtDNA maintenance gene SSBP1 cause optic atrophy and foveopathy

doi: 10.1172/JCI128513

Figure Lengend Snippet: ( A ) Crystal structure of SSBP1 showing that human SSBP1 consists of a dimer (molecules molA and molB) that contacts a second, symmetrically related dimer (molA′ and molB′), giving rise to the tetrameric biological unit. Both Arg38 and Arg107 are displayed in yellow (frame). ( B ) Zoom-in of mutated residues; Arg38 is replaced by Gln38 (top). The bottom panel is the same, except Arg107 is replaced by Gln 107. In each case, the mutated residue is shown in light gray. ( C ) SDS/PAGE analysis of SSBP1 monomers and dimers in control (control 1 and control 2) and patient fibroblasts (patient 1, patient 2, patient 3). Actin was used as a loading control. For each cell line, 3 independent biological replicates were loaded. ( D ) Densitometric quantification of SSBP1 monomer (left) and dimer (right) in pooled controls and patient fibroblasts. Data are shown as scatter plots with mean ± SEM indicated. ** P < 0.01; *** P < 0.001, 1-way ANOVA with Dunnett’s correction. Note that some of the same samples (for control 1, patient 1, and patient 2) were run on both membranes and these thus represent technical replicates. The ratios with actin from both membranes were averaged prior to statistical analysis.

Article Snippet: Membranes were saturated with 5% nonfat milk dissolved in 0.1% Tween-TBS for 2 hours at room temperature, then incubated overnight at 4°C with polyclonal sheep anti-SSBP1 (1 μg/ml, catalog AF6588, Bio-techne), monoclonal mouse anti-Grim19 (1:1000, catalog ab110240, Abcam), anti-SDHA (1:1000, catalog ab14715, Abcam), anti-UQCRCII (1:1000, catalog ab14745, Abcam), anti-MTCO1 (1:1000, catalog ab14705, Abcam), anti-GAPDH (1:2000, catalog G8795, MilliporeSigma), and polyclonal rabbit anti-ATP synthase (1:1000, catalog ab151229, Abcam).

Techniques: Residue, SDS Page

Journal: Brazilian Journal of Medical and Biological Research

Article Title: Down-regulation of single-stranded DNA-binding protein 1 expression induced by HCMV infection promotes lipid accumulation in cells

doi: 10.1590/1414-431X20176389

Figure Lengend Snippet: Transcripts that showed high homology to known genes.

Article Snippet: The membranes were blocked in 5% BSA, and then incubated with Polyclonal sheep anti-human SSBP1 antibodies (R&D System, USA) followed by the appropriate horseradish peroxidase-conjugated secondary antibodies.

Techniques: Binding Assay, Sequencing, Ubiquitin Proteomics, Immunopeptidomics

Journal: Brazilian Journal of Medical and Biological Research

Article Title: Down-regulation of single-stranded DNA-binding protein 1 expression induced by HCMV infection promotes lipid accumulation in cells

doi: 10.1590/1414-431X20176389

Figure Lengend Snippet: Human cytomegalovirus (HCMV) down-regulated the mRNA and protein expression of single-stranded DNA-binding protein (SSBP1). A , Expression levels of SSBP1 assayed by real-time PCR. B , Protein expression levels of SSBP1 assayed by western blot. Data are reported as means±SE, (n=4). *P<0.05 and **P<0.01 compared to time 0 (ANOVA).

Article Snippet: The membranes were blocked in 5% BSA, and then incubated with Polyclonal sheep anti-human SSBP1 antibodies (R&D System, USA) followed by the appropriate horseradish peroxidase-conjugated secondary antibodies.

Techniques: Expressing, Binding Assay, Real-time Polymerase Chain Reaction, Western Blot

Journal: Brazilian Journal of Medical and Biological Research

Article Title: Down-regulation of single-stranded DNA-binding protein 1 expression induced by HCMV infection promotes lipid accumulation in cells

doi: 10.1590/1414-431X20176389

Figure Lengend Snippet: Single-stranded DNA-binding protein (SSBP1)-expression vector or siRNA were transfected into human umbilical vein endothelial cells. A , mRNA expression levels of SSBP1 assayed by real-time PCR. B , Protein expression levels of SSBP1 assayed by western blot. Data are reported as means±SE, n=4. **P<0.01 vs control (ANOVA).

Article Snippet: The membranes were blocked in 5% BSA, and then incubated with Polyclonal sheep anti-human SSBP1 antibodies (R&D System, USA) followed by the appropriate horseradish peroxidase-conjugated secondary antibodies.

Techniques: Binding Assay, Expressing, Plasmid Preparation, Transfection, Real-time Polymerase Chain Reaction, Western Blot, Control

Journal: Brazilian Journal of Medical and Biological Research

Article Title: Down-regulation of single-stranded DNA-binding protein 1 expression induced by HCMV infection promotes lipid accumulation in cells

doi: 10.1590/1414-431X20176389

Figure Lengend Snippet: Single-stranded DNA-binding protein SSBP1 affected the expression levels of lipid metabolism-associated genes in human umbilical vein endothelial cells. The results of real-time PCR showed that over-expression of SSBP1 inhibited the expression of LDLR and SCARB, while knockdown of SSBP1 significantly promoted the mRNA expression of LDLR, HMGR, CETP and SCARB. LDLR: low-density lipoprotein receptor; SCARB: scavenger receptor B; HMGCR: HMG-CoA reductase; CETP: cholesteryl ester transfer protein. Data are reported as means±SE, n=4. *P<0.05 and **P<0.01 vs control (ANOVA).

Article Snippet: The membranes were blocked in 5% BSA, and then incubated with Polyclonal sheep anti-human SSBP1 antibodies (R&D System, USA) followed by the appropriate horseradish peroxidase-conjugated secondary antibodies.

Techniques: Binding Assay, Expressing, Real-time Polymerase Chain Reaction, Over Expression, Knockdown, Control

Journal: Brazilian Journal of Medical and Biological Research

Article Title: Down-regulation of single-stranded DNA-binding protein 1 expression induced by HCMV infection promotes lipid accumulation in cells

doi: 10.1590/1414-431X20176389

Figure Lengend Snippet: Single-stranded DNA-binding protein (SSBP1) affected the total cholesterol content. The results showed that cholesterol in the cells decreased significantly in SSBP1-expression cells ( A ) and increased significantly in SSBP1 knock-down cells in a time-dependent manner ( B ). Data are reported as means±SE, n=6. *P<0.05 and **P<0.01 (ANOVA).

Article Snippet: The membranes were blocked in 5% BSA, and then incubated with Polyclonal sheep anti-human SSBP1 antibodies (R&D System, USA) followed by the appropriate horseradish peroxidase-conjugated secondary antibodies.

Techniques: Binding Assay, Expressing, Knockdown