polyclonal rabbit anti-pmp70 antibody  (Thermo Fisher)

Journal: Biomolecules

Article Title: ABCD1 Transporter Deficiency Results in Altered Cholesterol Homeostasis

doi: 10.3390/biom13091333

Figure Lengend Snippet: Low interaction between peroxisomes and lipid droplets under cholesterol-loading conditions in both control and X-ALD fibroblasts. ( A ) Western blot analysis depicting various levels of stable ABCD1 protein and GAPDH for normalisation of loading in the cell lines used for the co-localisation study in standard RPMI medium. The samples are colour-coded according to their ABCD1 content (blue, >20%; orange, 5–20%; red, <5%). A cut in the image is indicated by a dashed line; for the full set and quantification of ABCD1 protein levels, see . ( B – D ) Control (n = 3) and X-ALD-derived fibroblasts (n = 7) were starved in LDM, loaded with 20 µg/mL cholesterol for 72 h and stained with BODIPY™ 493/503 (for LDs) and anti-PMP70 antibody (for peroxisomes). Representative ultra-high resolution confocal images of LDs (green) and peroxisomes (red) and their interactions (yellow) in cholesterol-treated cells (control C1 and X-ALD A9) Original images could be found in —original-images ( B ). The extent of interaction between LDs and peroxisomes is expressed as Manders’ co-localisation coefficients, M1 (% of green pixel with red component) and M2 (% of red pixel with green component) ( C ), one-way ANOVA with Sidak’s multiple comparisons test. Linear regression analysis of the relationship between M1 or M2 and the level of mutated ABCD1 protein in X-ALD cells revealed no significant correlation ( D ).

Article Snippet: For peroxisomal staining, cells were incubated overnight at 4 °C with a polyclonal rabbit anti-PMP70 antibody (1:1000, Invitrogen, Waltham, MA, USA), followed by incubation with secondary donkey anti-rabbit IgG Cy3 antibody (1:400, #IR715-165-150, Jackson, West Grove, PA, USA) for 1 h at room temperature.

Techniques: Western Blot, Derivative Assay, Staining

Journal: Molecular Neurodegeneration

Article Title: Age-dependent roles of peroxisomes in the hippocampus of a transgenic mouse model of Alzheimer’s disease

doi: 10.1186/1750-1326-8-8

Figure Lengend Snippet: PMP70 protein levels and distribution in the hippocampus of WT and Tg mice. ( a ) Densitometric values of PMP70 WB, obtained analyzing the hippocampal protein extracts of 3-, 6-, 9-, 12-, and 18-month-old WT and Tg mice. Data are expressed as mean ± SD. *P <0.05; **P <0.01; ***P <0.001. ( b ) Immunohistochemical distribution of PMP70 in CA1 hippocampal field of 3-, 6-, 9-, and 18-month-old WT and Tg mice. or, stratum oriens; pyr, stratum pyramidale; rad, stratum radiatum. Scale bars, 25 μm. ( c ) PMP70 pre-embedding immunoelectron microscopy of 3-month-old CA1 pyramidal neurons of WT and Tg animals. In both genotypes positive peroxisomes (arrows) are observed and they appear more numerous in the cytoplasm of Tg neurons. N, neuronal nucleus; m, mitochondrion. Scale bars, 1 μm.

Article Snippet: Sections were incubated overnight at 4°C with a mixture of rabbit polyclonal anti-PMP70 (1:100, Sigma-Aldrich) and either mouse monoclonal anti-GFAP (1:200, Chemicon, Temecula, CA, USA), or mouse monoclonal anti-Neuronal Nuclei (anti-NeuN 1:100, Chemicon).

Techniques: Immunohistochemical staining, Immuno-Electron Microscopy

Journal: Molecular Neurodegeneration

Article Title: Age-dependent roles of peroxisomes in the hippocampus of a transgenic mouse model of Alzheimer’s disease

doi: 10.1186/1750-1326-8-8

Figure Lengend Snippet: GFAP protein levels and distribution in the hippocampus of WT and Tg mice. ( a ) GFAP immunohistochemical localization in CA1 hippocampal field of 9-, 12-, and 18-month-old WT and Tg mice . or, stratum oriens; pyr, stratum pyramidale; rad, stratum radiatum. Scale bars, 25 μm. ( b ) Densitometric analysis of GFAP WB performed on hippocampal protein extracts of 3-, 6-, 9-, 12-, and 18-month-old mice. Values are expressed as mean ± SD. **P < 0.01; ***P < 0.001. ( c ) Double immunofluorescence of PMP70 (green) in combination with GFAP (red) in the CA1 hippocampal field of 18-month-old WT and Tg brain. Several PMP70 + /GFAP + cells (yellow) are especially numerous in the pathological genotype. Scale bars, 25 μm.

Article Snippet: Sections were incubated overnight at 4°C with a mixture of rabbit polyclonal anti-PMP70 (1:100, Sigma-Aldrich) and either mouse monoclonal anti-GFAP (1:200, Chemicon, Temecula, CA, USA), or mouse monoclonal anti-Neuronal Nuclei (anti-NeuN 1:100, Chemicon).

Techniques: Immunohistochemical staining, Immunofluorescence

Journal: bioRxiv

Article Title: Pkd1 mutation has no apparent effects on peroxisome structure or lipid metabolism

doi: 10.1101/2021.02.08.430145

Figure Lengend Snippet: a) A schematic diagram of the current model for de novo formation of peroxisomes showing the hybrid origin of peroxisomes: nascent peroxisome vesicles derived from mitochondria (PEX3) and ER (PEX16 and other peroxins) fuse and then undergo subsequent stepwise assembly of peroxisome components to form mature peroxisomes. The functional peroxisomes interplay with mitochondria in metabolic cellular processes such as fatty acid oxidation. b) Representative images of Pkd1 control and mutant kidney epithelial cells (121112CLTL cell line) double immunostained for PMP70 and catalase. Peroxisomal number, size and distribution were similar in Pkd1 mutant and control cells both at 37 °C (the images in the top two rows) and after 48 hours incubation at 40 °C (the images in the lower two rows). Scale bar, 10μm. c) Boxplots showing quantification of the number of PMP70/catalase double positive peroxisomes in Pkd1 mutant and control cells derived from two kidney epithelial cell lines (121112CLTL, 96784LTL) cultured at 37°C and at 40 °C. None of the cell lines manifest a significant decrease in peroxisomal number at high temperature (40°C). d, e) Boxplots show quantification of peroxisomal area (d) and distance between PMP70 positive peroxisomes to nucleus (e) in the 121112CLTL and 96784LTL kidney cell lines.

Article Snippet: The cells were incubated with an Odyssey Blocking Buffer (LI-COR, 927-40000) for 30 minutes, then incubated with a rabbit polyclonal anti-PMP70 (Thermo Fisher Scientific, PA1-650, 1: 200), a goat anticatalase (R&D systems, AF3398, 1: 150), a rabbit anti-Tom20 (Santa Cruz Biotechnology, FL-145, 1: 100), or a mouse anti-HA (MBL, M180-3, 1: 200) antibody followed by incubation with fluorescently labeled secondary antibodies (Thermo Fisher Scientific, [A-31572, 1:1000], [A-27034, 1:1000], [A-28180, 1:1000], [A-32814, 1:1000]).

Techniques: Derivative Assay, Functional Assay, Mutagenesis, Incubation, Cell Culture

Journal: bioRxiv

Article Title: Pkd1 mutation has no apparent effects on peroxisome structure or lipid metabolism

doi: 10.1101/2021.02.08.430145

Figure Lengend Snippet: a, b) Representative immunoblot of protein lysates from three different wild type ( Pkd1+ ) and mutant ( Pkd1- ) pairs of kidney cell lines probed for peroxisomal associated proteins PMP70 (a), Pex3 and Pex5 (b). α-tubulin was used as a loading control. b) Quantification of the data from the immunoblot studies shows comparable levels of protein expression in Pkd1 control and mutant cells. The signal intensity values of PMP70, Pex3 and Pex5 values were individually normalized to the intensity of the α-tubulin band detected in the same lane. The results for three separate studies of each cell line pair are shown.

Article Snippet: The cells were incubated with an Odyssey Blocking Buffer (LI-COR, 927-40000) for 30 minutes, then incubated with a rabbit polyclonal anti-PMP70 (Thermo Fisher Scientific, PA1-650, 1: 200), a goat anticatalase (R&D systems, AF3398, 1: 150), a rabbit anti-Tom20 (Santa Cruz Biotechnology, FL-145, 1: 100), or a mouse anti-HA (MBL, M180-3, 1: 200) antibody followed by incubation with fluorescently labeled secondary antibodies (Thermo Fisher Scientific, [A-31572, 1:1000], [A-27034, 1:1000], [A-28180, 1:1000], [A-32814, 1:1000]).

Techniques: Western Blot, Mutagenesis, Expressing

Journal: Veterinary microbiology

Article Title: Porcine deltacoronavirus (PDCoV) infection antagonizes interferon-λ1 production.

doi: 10.1016/j.vetmic.2020.108785

Figure Lengend Snippet: Fig. 5. PDCoV infection decreases the number of peroxisomes and inhibits IRF1 nuclear translocation. (A) IPI-2I cells were infected with PDCoV at a MOI of 0.5. At the indicated time points after infection, cells were collected and subjected to western blotting with antibodies against IRF1, MAVS and PDCoV N protein. β-actin was used as a control for sample loading. (B) IPI-2I cells were infected with PDCoV for 24 h or stimulated with SeV for 12 h as a positive control, and then fixed for indirect immunofluorescence assays with antibodies against PMP70 (peroxisome marker) and PDCoV N protein. Peroxisomes and PDCoV N protein were visualized by Alexa Fluor 488-conjugated donkey anti-mouse (green) and 594-conjugated donkey anti-rabbit (red) antibodies, respectively. Cellular nuclei were counterstained with 1 μg/mL of DAPI. Fluorescence was observed under a Fluoview ver. 3.1 confocal fluorescence microscope (Olympus) and representative images are shown. (C) IPI-2I cells were mock-infected or infected with PDCoV (MOI = 0.5) for 24 h or stimulated with SeV for 12 h as a positive control. The cells were fixed and the number of peroxisomes in PDCoV-infected cells, mock-infected cells or cells stimulated with SeV was quantified. The data are presented as the means ± SD of three independent experiments (*p < 0.05). (D) IPI-2I cells were infected with PDCoV (MOI = 0.5) for 12 h followed by stimulation with SeV for 12 h. Cells were fixed and incubated with anti-PDCoV N protein antibody and anti-IRF1 antibody for 1 h. IRF1 and PDCoV N were visualized by Alexa Fluor 488-conjugated (green) donkey anti-mouse and 594-conjugated (red) donkey anti-rabbit antibodies. Cellular nuclei were counterstained with DAPI. White arrows indicate the nucleus-localized IRF1 and yellow arrows indicate the cytoplasm-localized IRF1 (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article).

Article Snippet: Rabbit anti-IRF1 polyclonal antibody (Bioss Antibodies, bs21318R) and rabbit anti-PMP70 polyclonal antibody (Novus Biologicals, NBP187258) were used for immunofluorescence assays (IFAs) and western blotting.

Techniques: Infection, Translocation Assay, Western Blot, Control, Positive Control, Marker, Fluorescence, Microscopy, Incubation