polyclonal rabbit tomm20 antibody  (Santa Cruz Biotechnology)

Journal: Nature Communications

Article Title: Automatic optimization of flat-field corrections by evaluation and enhancement (EVEN) in multimodal optical microscopy

doi: 10.1038/s41467-025-68150-0

Figure Lengend Snippet: A three-channel fluorescence microscopy measurement of stained HEK293 cells measured by Ph2 objective is automatically optimized by EVEN (prediction dataset 2, red: peroxisomal proteins (anti-GFP nanobody); green: TOMM20 protein; blue: peroxisomal proteins (eGFP)). a Raw multi-channel image. The inset shows the 2 × 2 tile section of the image used in this figure, with dashed white lines marking tile borders. Multiple corrections are obtained by applying BaSiC, CIDRE, Fourier methods, and then optimizing the multi-channel image with EVEN. EVEN selects CIDRE for the red and green channel, and Fourier for the blue channel. b Steps to analyse the measurements of stained cells: multi-channel images are converted to greyscale by summing the single channels (that contain signals from different components of the cytoplasm) and are analysed with automatic cells segmentation using Cellpose . The greyscale image is obtained for the raw measurement, the single-channel corrections and the EVEN optimization. c Intensity sum (along y) of the greyscale inset for each method. The black dashed line indicates the border between neighbouring tiles. The corrected images show higher intensities at the edges of the tiles and the enhancement of sample features. EVEN and CIDRE show the greatest intensity recovery between tiles. d Top row: multi-channel images obtained with single-method corrections and EVEN optimization; the white dashed boxes highlight two regions significantly improved by EVEN. Bottom row: Cellpose prediction on the greyscale sum of the three channels for each method. After correction of uneven illumination, Cellpose can outline a greater number of cells, especially at the borders of neighbouring tiles. White dashed boxes highlight three regions where EVEN optimization provides better identification of the cells compared to non-optimized images. Bottom labels show, for each image, the normalized EVEN score summed over three channels and the cell count in the zoomed region. While counts are not strictly correlated with segmentation performance, good correction of uneven illumination enhances downstream analysis and generally increases the number of detected cells. Further quantification is provided in Supplementary Fig. . Scale bar: 180 µm, size of a single tile.

Article Snippet: Additionally, immunolabeling was performed on TOMM20-protein with anti-Tomm20 rabbit polyclonal antibodies (proteintech, USA), dilution 1:200, and goat anti-rabbit IgG secondary antibodies labelled with Abberior STAR Orange (Abberior, Germany) at a dilution of 1:350.

Techniques: Fluorescence, Microscopy, Staining, Cell Counting

Journal: Cell Death & Disease

Article Title: PBLD promotes IRF3 mediated the type I interferon (IFN-I) response and apoptosis to inhibit viral replication

doi: 10.1038/s41419-024-07083-w

Figure Lengend Snippet: A HeLa cells were transfected with Flag-PBLD or pCMV-Flag for 24 h, and then the cells were infected with BPIV3 (MOI = 1) for the indicated time. The expression of intrinsic mitochondrial apoptosis proteins was determined by western blot analysis. B , C HeLa cells were transfected with pCMV-Flag, pCMV-HA-IRF3, siIRF3, or siNC for 24 h, and then the cells were infected with BPIV3 (MOI = 1) for the indicated time. The expression of intrinsic mitochondrial apoptosis proteins was determined by western blot analysis. D Flag-PBLD HeLa cell lines were treated with siIRF3 or scrambled siRNA (siNC) for 24 h, and then infected with BPIV3 (MOI = 1) for the indicated time. The expression of Bax, Bcl 2 , Cytc, and cleaved PARP proteins were analyzed by western blotting. E Mitochondrial isolation and sub-cellular localization assay for IRF3 affected by PBLD during BPIV3 infection. The cells were transfected with Flag-PBLD or vector for 24 h. Following BPIV3 (MOI = 1) infection for 24 h, cytosolic and mitochondrial proteins were prepared, and the expression of IRF3 was determined by western blot analysis. β-actin and TOMM20 were used for loading control of cytosolic and mitochondrial protein, respectively. F Mapping the ubiquitination sites of IRF3. G – J pCMV-Flag or pCMV-Flag-PBLD in combination of pCMV-HA-IRF3, pCMV-HA-IRF3(S385/386 A) and pCMV-HA-IRF3(S396A) plasmids were transfected into IRF3 knockout HeLa cell lines and then infected with BPIV3 (MOI = 1) or SeV (100HAU/mL) for 24 h, respectively. The expression of cleaved PARP apoptosis protein was verified by western blotting. The gray intensity of the bands in ( A – E and G – J ) from three independent experiments were analyzed using ImageJ software. Significance was determined by two-way ANOVA in ( A – E ), one-way ANOVA in ( G – J ). Ns not significant, * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: Rabbit polyclonal antibody to TOMM20 (#AF5206) was obtained from Affinity Biosciences.

Techniques: Transfection, Infection, Expressing, Western Blot, Isolation, Plasmid Preparation, Control, Knock-Out, Software

Journal: Cell Death & Disease

Article Title: PBLD promotes IRF3 mediated the type I interferon (IFN-I) response and apoptosis to inhibit viral replication

doi: 10.1038/s41419-024-07083-w

Figure Lengend Snippet: A Interaction between IRF3 and Puma was analyzed by co-immunoprecipitation. HeLa cells expressing HA-IRF3 were exposed to BPIV3 infection for 24 h, and then immunoprecipitation of IRF3 was performed, and the presence of Puma and Noxa proteins was assessed by western blot analysis. B The effect of PBLD on the binding between IRF3 and Puma was examined by co-immunoprecipitation analysis. HeLa cells were cotransfected with HA-IRF3 and different doses (1.2 μg, 2.4 μg) of PBLD for 24 h, and then infected with BPIV3 for another 24 h. After that, the cell lysates were immunoprecipitated with HA antibody, and the presence of Puma was detected by western blot analysis. C PBLD facilitates the interaction between IRF3 and Puma within the mitochondria as demonstrated by western blot analysis. HeLa cells were transfected with Flag-PBLD and infected with BPIV3 for 24 h. The mitochondrial and cytosolic fractions were isolated and analyzed by western blot for the indicated proteins. β-actin and TOMM20 function as the loading control for cytosolic proteins and mitochondrial proteins, respectively. D PBLD promoted the localization of IRF3 at the mitochondria and was examined in Puma knockout cell lines. E The effect of PBLD on the mitochondrial localization of Puma was detected in IRF3 knockout cell lines. F Wild-type IRF3 (HA-IRF3) and IRF3 mutants: HA-IRF3(K313/315 N) interacted with Puma was analyzed by co-immunoprecipitation in the context of PBLD. G – J Knockout of Puma attenuated IRF3- or PBLD-induced apoptosis during BPIV3 or VSV infection. Puma knockout cell lines were transfected with HA-IRF3 or Flag-PBLD, and infected with BPIV3 at a MOI of 1 for the indicated time. Then, apoptosis-associated proteins in these cells were determined by western blotting. The gray intensity of the bands in ( A – J ) from three independent experiments was analyzed using ImageJ software. Significance was determined by Student’s t -test in ( A ), one-way ANOVA in ( B ), two-way ANOVA in ( C – J ). Ns, not significant, * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: Rabbit polyclonal antibody to TOMM20 (#AF5206) was obtained from Affinity Biosciences.

Techniques: Immunoprecipitation, Expressing, Infection, Western Blot, Binding Assay, Transfection, Isolation, Control, Knock-Out, Software

Journal: Frontiers in Genetics

Article Title: Severe mitochondrial encephalomyopathy caused by de novo variants in OPA1 gene

doi: 10.3389/fgene.2024.1437959

Figure Lengend Snippet: Transcript level and protein amount of OPA1 in patients’ fibroblasts. (A) Expression level of OPA1 transcript; (B) Western blot of OPA1, a representative experiment out of three is shown; TOMM20 was used as loading control. (C,D) Densitometric analysis of OPA1/TOMM20 and OPA1 long/short forms ratio. Data from patients’ fibroblasts (Pt1, Pt2) were compared to the mean value of controls (=1) and are expressed as mean ± SEM (n = 3).

Article Snippet: The primary polyclonal rabbit TOMM20 antibody (Santa Cruz Biotechnology) was incubated in 4% bovine serum albumin in PBS overnight at 4°C.

Techniques: Expressing, Western Blot, Control

Journal: Frontiers in Genetics

Article Title: Severe mitochondrial encephalomyopathy caused by de novo variants in OPA1 gene

doi: 10.3389/fgene.2024.1437959

Figure Lengend Snippet: Impact of OPA1 defect on mtDNA amount and mitochondrial complex (MC) subunits level in patients’ fibroblasts. (A) Quantification of mtDNA amount in fibroblasts from patients (Pt1, Pt2) and controls (Ctrls). The amount of mtDNA was normalized to nuclear DNA and compared to the mean value of controls (=1). Two different probes for mtDNA were used ( ND1 , ND4 ). Data are represented as mean ± SEM of Ctrls (n = 10) and patients (n = 3) **: p < 0.005 in Mann-Whitney test. (B) Western blot of mitochondrial complexes subunits, a representative experiment out of three is shown; TOMM20 was used as loading control. (C) Densitometric analysis of mitochondrial complexes subunits/TOMM20. Data from patients’ fibroblasts (Pt1, Pt2) were compared to the mean value of controls (=1) and are expressed as mean ± SEM (n = 4). **: p < 0.005 in Mann-Whitney test.

Article Snippet: The primary polyclonal rabbit TOMM20 antibody (Santa Cruz Biotechnology) was incubated in 4% bovine serum albumin in PBS overnight at 4°C.

Techniques: MANN-WHITNEY, Western Blot, Control

Journal: Frontiers in Genetics

Article Title: Severe mitochondrial encephalomyopathy caused by de novo variants in OPA1 gene

doi: 10.3389/fgene.2024.1437959

Figure Lengend Snippet: Mitochondrial morphology in patients’ fibroblasts. Representative images of mitochondrial morphology in fibroblasts from control (Ctrl) and patients (Pt1, Pt2), labeled with TOMM20 antibody. Cells were grown either in regular medium (RM) (upper panel) or galactose medium (GAL) for 48 h (lower panel). Scale bar: 30 μm.

Article Snippet: The primary polyclonal rabbit TOMM20 antibody (Santa Cruz Biotechnology) was incubated in 4% bovine serum albumin in PBS overnight at 4°C.

Techniques: Control, Labeling