polyclonal rabbit antibodies against tom20  (Santa Cruz Biotechnology)

Journal: Scientific reports

Article Title: Uncovering the impact of UV radiation on mitochondria in dermal cells: a STED nanoscopy study.

doi: 10.1038/s41598-024-55778-z

Figure Lengend Snippet: Figure 1. UV irradiation induces the distribution change of mitochondrial membrane proteins. (a) At each condition, the cells were fixed and labeled with anti-TOM20 antibody. Images were obtained by confocal and STED microscopy. (b) The density of TOM20 clusters was quantitatively analyzed in each region of interest (ROI). Ten ROIs were included in each group for generating quantitative analysis plots. The data are depicted as mean ± SD. ****p < 0.0001.

Article Snippet: Polyclonal rabbit antibodies against TOM20 from Santa Crus Biotechnology and polyclonal mouse antibody against TFAM from Abnova were used as primary antibodies for labeling mitochondrial proteins.

Techniques: Irradiation, Membrane, Labeling, Microscopy

Journal: Scientific reports

Article Title: Uncovering the impact of UV radiation on mitochondria in dermal cells: a STED nanoscopy study.

doi: 10.1038/s41598-024-55778-z

Figure Lengend Snippet: Figure 3. Quantitative analysis of TOM20 clusters on mice skin tissues using STED nanoscopy. (a) UV irradiation was applied three times a week for 8 weeks to induce chronic UV damaged skin (photoaged mice skin model). Dorsal back skin was harvested 72 h after the last UV irradiation. Skin tissues were fixed and then paraffin embedded for further analysis. Immunofluorescence staining was performed using anti-TOM20 antibody. (b) Epidermal layer, the outermost compartment of the skin, was closely examined through both confocal and STED microscopy (c) TOM20 cluster densities can be quantitatively analyzed from STED images. (n = 5–6 mice) *** p < 0.0002.

Article Snippet: Polyclonal rabbit antibodies against TOM20 from Santa Crus Biotechnology and polyclonal mouse antibody against TFAM from Abnova were used as primary antibodies for labeling mitochondrial proteins.

Techniques: Irradiation, Immunofluorescence, Staining, Microscopy

Journal: Physiological Reports

Article Title: TOM complex‐independent transport pathway of myoglobin into mitochondria in C2C12 myotubes

doi: 10.14814/phy2.15632

Figure Lengend Snippet: Mb interacts with TOM complex receptors, Tom20 and Tom70. IP analysis was performed using mitochondria isolated from C2C12 myotubes on differentiation day 5. Fifty micrograms of protein from the mitochondrial fraction were immunoprecipitated with anti‐Tom20, anti‐Tom70, or anti‐IgG rabbit antibody. Subsequently, immunoblotting was performed with anti‐Mb, anti‐VDAC, anti‐Cyt c , anti‐Tom20 (a), or anti‐Tom70 mouse antibody (b). In every assay, 2.5 μg of the mitochondrial fraction from C2C12 myotubes on differentiation day 5 was used as a positive control (CON). Immunoprecipitation of normal rabbit IgG was used as a negative control. Unprocessed blots are available in Figure . CON, control; Cyt c , cytochrome c ; IgG, immunoglobulin G; IP, immunoprecipitation; Mb, myoglobin; Tom20, translocase of outer membrane 20; Tom70, translocase of outer membrane 70; VDAC; voltage‐dependent anion channel.

Article Snippet: After blocking, the membranes were washed three times with TBS‐T for 10 min, and then incubated with mouse monoclonal antibodies against α‐tubulin (1:20,000; 66031‐1‐Ig; Proteintech), cytochrome c (Cyt c ; 1:5,000; 66264‐1‐Ig; Proteintech), oxidative phosphorylation complexes (1:1,000; ab110413; abcam), Mb (1:1,000; sc‐393020; Santa Cruz Biotechnology), Tom70 (1:5,000; 66593‐1‐Ig; Proteintech), and apoptosis‐inducing factor (AIF; 1:1,000; sc‐55519; Santa Cruz Biotechnology), and rabbit polyclonal antibodies against Tom20 (1:5,000; 11802‐1‐AP; Proteintech), voltage‐dependent anion channel (VDAC; 1:5,000; 55259‐1‐AP; Proteintech), Tom40 (1:5,000; 18409‐1‐AP; Proteintech), mitochondrial heat shock protein 70 (mtHSP70; 1:5,000; 14887‐1‐AP; Proteintech), COX‐IV (1:1,000; 11242‐1‐AP; Proteintech), and PTEN‐induced kinase 1 (PINK1; 1:1,000; 23274‐1‐AP; Proteintech) overnight at 4°C.

Techniques: Isolation, Immunoprecipitation, Western Blot, Positive Control, Negative Control, Control, Membrane

Journal: Physiological Reports

Article Title: TOM complex‐independent transport pathway of myoglobin into mitochondria in C2C12 myotubes

doi: 10.14814/phy2.15632

Figure Lengend Snippet: Mb is imported into mitochondria in the absence of Tom20. (a) CON‐siRNA or Tom20‐siRNA‐transfected myoblasts were differentiated into myotubes following incubation in differentiation medium for 3 days. Scale bar shows 200 μm. (b) Mitochondria were isolated from C2C12 myotubes transfected with Tom20‐siRNA or CON‐siRNA. Mitochondrial fractions were immunoblotted for Tom20, COX‐IV, Mb, and AIF. (c) Quantification of the immunoreactivities of siRNA‐target protein Tom20 and COX‐IV (Tom20‐dependent import) ( n = 3/group). (d) Quantification of the immunoreactivities of Mb and AIF (TOM complex‐independent import) ( n = 3/group). All calculated data were normalized to CBB staining. The mean immunoreactivity of proteins in CON‐siRNA‐transfected cells was set to 100%. Values are means ± standard deviation. Significant differences were assessed using an unpaired t‐ test. * indicates significantly different from CON‐siRNA‐transfected cells ( p < 0.05). Unprocessed blots are available in Figure . AIF, apoptosis‐inducing factor; CBB, Coomassie brilliant blue; CON, control; COX‐IV, cytochrome c oxidase subunit IV; Mb, myoglobin; TOM, translocase of the outer membrane; Tom20, translocase of outer membrane 20.

Article Snippet: After blocking, the membranes were washed three times with TBS‐T for 10 min, and then incubated with mouse monoclonal antibodies against α‐tubulin (1:20,000; 66031‐1‐Ig; Proteintech), cytochrome c (Cyt c ; 1:5,000; 66264‐1‐Ig; Proteintech), oxidative phosphorylation complexes (1:1,000; ab110413; abcam), Mb (1:1,000; sc‐393020; Santa Cruz Biotechnology), Tom70 (1:5,000; 66593‐1‐Ig; Proteintech), and apoptosis‐inducing factor (AIF; 1:1,000; sc‐55519; Santa Cruz Biotechnology), and rabbit polyclonal antibodies against Tom20 (1:5,000; 11802‐1‐AP; Proteintech), voltage‐dependent anion channel (VDAC; 1:5,000; 55259‐1‐AP; Proteintech), Tom40 (1:5,000; 18409‐1‐AP; Proteintech), mitochondrial heat shock protein 70 (mtHSP70; 1:5,000; 14887‐1‐AP; Proteintech), COX‐IV (1:1,000; 11242‐1‐AP; Proteintech), and PTEN‐induced kinase 1 (PINK1; 1:1,000; 23274‐1‐AP; Proteintech) overnight at 4°C.

Techniques: Transfection, Incubation, Isolation, Staining, Standard Deviation, Control, Membrane

Journal: Physiological Reports

Article Title: TOM complex‐independent transport pathway of myoglobin into mitochondria in C2C12 myotubes

doi: 10.14814/phy2.15632

Figure Lengend Snippet: Mb is imported into mitochondria in the absence of Tom70. (a) CON‐siRNA or Tom70‐siRNA‐transfected myoblasts were differentiated into myotubes following incubation in differentiation medium for 3 days. Scale bar shows 200 μm. (b) Mitochondria were isolated from C2C12 myotubes transfected with Tom70‐siRNA or CON‐siRNA. Mitochondrial fractions were immunoblotted for Tom70, PINK1, Mb, and AIF. (c) Quantification of the immunoreactivities of siRNA‐target protein Tom70 and PINK1 (Tom70‐dependent import) ( n = 3/group). (d) Quantification of the immunoreactivities of Mb, COX‐IV (Tom20‐dependent import) and AIF (TOM complex‐independent import) ( n = 3/group). All calculated data were normalized to CBB staining. The mean immunoreactivity of proteins in CON‐siRNA‐transfected cells was set to 100%. Values are means ± standard deviation. Significant differences were assessed using an unpaired t‐ test. * indicates significantly different from CON‐siRNA‐transfected cells ( p < 0.05). Unprocessed blots are available in Figure . AIF, apoptosis‐inducing factor; CBB, Coomassie brilliant blue; CON, control; Mb, myoglobin; PINK1, PTEN‐induced kinase 1; TOM, translocase of the outer membrane; Tom70, translocase of outer membrane 70.

Article Snippet: After blocking, the membranes were washed three times with TBS‐T for 10 min, and then incubated with mouse monoclonal antibodies against α‐tubulin (1:20,000; 66031‐1‐Ig; Proteintech), cytochrome c (Cyt c ; 1:5,000; 66264‐1‐Ig; Proteintech), oxidative phosphorylation complexes (1:1,000; ab110413; abcam), Mb (1:1,000; sc‐393020; Santa Cruz Biotechnology), Tom70 (1:5,000; 66593‐1‐Ig; Proteintech), and apoptosis‐inducing factor (AIF; 1:1,000; sc‐55519; Santa Cruz Biotechnology), and rabbit polyclonal antibodies against Tom20 (1:5,000; 11802‐1‐AP; Proteintech), voltage‐dependent anion channel (VDAC; 1:5,000; 55259‐1‐AP; Proteintech), Tom40 (1:5,000; 18409‐1‐AP; Proteintech), mitochondrial heat shock protein 70 (mtHSP70; 1:5,000; 14887‐1‐AP; Proteintech), COX‐IV (1:1,000; 11242‐1‐AP; Proteintech), and PTEN‐induced kinase 1 (PINK1; 1:1,000; 23274‐1‐AP; Proteintech) overnight at 4°C.

Techniques: Transfection, Incubation, Isolation, Staining, Standard Deviation, Control, Membrane

Journal: Physiological Reports

Article Title: TOM complex‐independent transport pathway of myoglobin into mitochondria in C2C12 myotubes

doi: 10.14814/phy2.15632

Figure Lengend Snippet: Mb is imported into mitochondria in the absence of Tom20 and Tom70. (a) CON‐siRNA or Tom20‐siRNA and Tom70‐siRNA double transfected myoblasts were differentiated into myotubes following incubation in differentiation medium for 3 days. Scale bar shows 200 μm. (b) Mitochondria were isolated from C2C12 myotubes transfected with Tom20‐siRNA and Tom70‐siRNA or CON‐siRNA. Mitochondrial fractions were immunoblotted for Tom20, Tom70, COX‐IV, PINK1, Mb, and AIF. (c) Quantification of the immunoreactivities of siRNA‐target protein Tom20 and Tom70, COX‐IV and PINK1 (Tom20‐ and Tom70‐dependent import, respectively) ( n = 3/group). (d) Quantification of the immunoreactivities of Mb and AIF (TOM complex‐independent import) ( n = 3/group). All calculated data were normalized to CBB staining. The mean immunoreactivity of proteins in CON‐siRNA‐transfected cells was set to 100%. Values are means ± standard deviation. Significant differences were assessed using an unpaired t‐ test. * indicates significantly different from CON‐siRNA‐transfected cells ( p < 0.05). Unprocessed blots are available in Figure . AIF, apoptosis‐inducing factor; CBB, Coomassie brilliant blue; CON, control; COX‐IV, cytochrome c oxidase subunit IV; Mb, myoglobin; PINK1, PTEN‐induced kinase 1; TOM, translocase of the outer membrane; Tom20, translocase of outer membrane 20; Tom70, translocase of outer membrane 70.

Article Snippet: After blocking, the membranes were washed three times with TBS‐T for 10 min, and then incubated with mouse monoclonal antibodies against α‐tubulin (1:20,000; 66031‐1‐Ig; Proteintech), cytochrome c (Cyt c ; 1:5,000; 66264‐1‐Ig; Proteintech), oxidative phosphorylation complexes (1:1,000; ab110413; abcam), Mb (1:1,000; sc‐393020; Santa Cruz Biotechnology), Tom70 (1:5,000; 66593‐1‐Ig; Proteintech), and apoptosis‐inducing factor (AIF; 1:1,000; sc‐55519; Santa Cruz Biotechnology), and rabbit polyclonal antibodies against Tom20 (1:5,000; 11802‐1‐AP; Proteintech), voltage‐dependent anion channel (VDAC; 1:5,000; 55259‐1‐AP; Proteintech), Tom40 (1:5,000; 18409‐1‐AP; Proteintech), mitochondrial heat shock protein 70 (mtHSP70; 1:5,000; 14887‐1‐AP; Proteintech), COX‐IV (1:1,000; 11242‐1‐AP; Proteintech), and PTEN‐induced kinase 1 (PINK1; 1:1,000; 23274‐1‐AP; Proteintech) overnight at 4°C.

Techniques: Transfection, Incubation, Isolation, Staining, Standard Deviation, Control, Membrane

Journal: The Journal of biological chemistry

Article Title: Labeling and measuring stressed mitochondria using a PINK1-based ratiometric fluorescent sensor.

doi: 10.1016/j.jbc.2021.101279

Figure Lengend Snippet: Figure 1. Development of a novel sensor to detect mitochondrial stress. A, schematic representation of mito-Pain F. Numbers above the construct indicate amino acid residues. B, a schematic diagram of the measurement of mitochondrial stress by mito-Pain. C, the T2A peptide sequence did not interfere with the localization of PINK1-GFP. HeLa cells stably expressing mito-Pain F or PINK1-GFP were cultured with CCCP for 24 h before fixation. The cells were stained with an antibody against Tom20 and observed by fluorescence microscopy. The scale bar represents 5 μm. The inset scale bar represents 1 μm. D, mito-Pain F is efficiently cleaved at the T2A site. HeLa cells and HeLa cells expressing mito-Pain F or PINK1-GFP were treated with CCCP or DMSO for 24 h and analyzed by immunoblotting using antibodies against PINK1, GFP, and RFP. Asterisks indicate nonspecific bands. E, PINK1-GFP, but not RFP-Omp25, of mito-Pain F was significantly increased under mitochondrial depolarization. HeLa cells stably expressing mito-Pain F were cultured with CCCP or DMSO for 24 h and then analyzed by flow cytometry. Cells for fluorescence microscopy were subjected to the same treatment and then fixed. The data represent the mean (left bottom panel: GFP-RFP fluorescence ratio) (n = 9). The main scale bar represents 5 μm. The inset scale bar represents 1 μm. CCCP, carbonyl cyanide m-chlorophenyl hydrazone; DMSO, dimethyl sulfoxide; mito-Pain, mitochondrial PINK1 accumulation index; mito-Pain F, mitochondrial PINK1 accumulation index (containing full-length PINK1); MTS, mitochondrial targeting sequence; OMS, outer mitochondrial membrane localization signal; PINK1, PTEN-induced putative kinase 1; TMD, transmembrane domain.

Article Snippet: A rabbit polyclonal antibody against Tom20 (code No. sc-11415) was purchased from Santa Cruz Biotechnology.

Techniques: Construct, Sequencing, Stable Transfection, Expressing, Cell Culture, Staining, Microscopy, Western Blot, Cytometry, Membrane

Journal: The Journal of biological chemistry

Article Title: Labeling and measuring stressed mitochondria using a PINK1-based ratiometric fluorescent sensor.

doi: 10.1016/j.jbc.2021.101279

Figure Lengend Snippet: Figure 2. Improvement of mito-Pain by removing the PINK1 kinase domain. A, schematic representation of mito-Pain T (PINK1 truncated). Numbers above the construct indicate amino acid residues of each protein (the PB1 domain from PKCζ and the degron domain from Rpn4.) B, mito-Pain T showed a much higher signal ratio than mito-Pain F. Polyclonal HeLa cells stably expressing mito-Pain T were cultured with CCCP or DMSO for 24 h and then analyzed by flow cytometry. Cells for fluorescence microscopy were subjected to the same treatment and then fixed. The data represent the mean (right panel: GFP-RFP fluorescence ratio) (n = 7). The scale bar represents 5 μm. The inset scale bar represents 1 μm. C, graph showing the results of quantification of GFP-RFP fluorescence ratios (n = 9 for mito-Pain F and n = 7 for mito-Pain T). The data represent the mean ± SD. D, high expression of mito-Pain T–induced mitochondrial aggregation. HeLa cells highly expressing mito-Pain T were cultured with CCCP for 24 h before fixation. The scale bar represents 5 μm. The inset scale bar represents 1 μm. E, the GFP signal of mito-Pain T mainly localized to mitochondria under mitochondrial depolarization. HeLa cells expressing mito-Pain T were cultured with CCCP for 24 h before fixation. The cells were stained with antibodies against Tom20, Trapα, GM130, Lamp1, or Pex14 and observed by fluorescence microscopy. The scale bar represents 5 μm. The inset scale bar represents 1 μm. F, detection of several mitochondrial stresses using mito-Pain T. HeLa cells expressing mito-Pain T were cultured with the indicated reagent for 24 h and then analyzed by flow cytometry. The value obtained by dividing the GFP/RFP of the compound treatment by GFP/RFP of the DMSO treatment is called PNIK1 stability. The data represent the mean ± SD (n = 3). *p < 0.05 and **p < 0.01. G, mito-Pain can distinguish cytoplasmic and mitochondrial accumulated PINK1-GFP. HeLa cells expressing mito-Pain T were cultured with MG-132 or DMSO for 6 h before fixation. The scale bar represents 5 μm. CCCP, carbonyl cyanide m-chlorophenyl hydrazone; DMSO, dimethyl sulfoxide; mito-Pain, mitochondrial PINK1 accumulation index; mito-Pain F, mitochondrial PINK1 accumulation index (containing full-length PINK1); mito-Pain T, truncated mito-Pain; MTS, mitochondrial targeting sequence; PINK1, PTEN-induced putative kinase 1; TMD, transmembrane domain.

Article Snippet: A rabbit polyclonal antibody against Tom20 (code No. sc-11415) was purchased from Santa Cruz Biotechnology.

Techniques: Construct, Stable Transfection, Expressing, Cell Culture, Cytometry, Microscopy, Staining, Sequencing