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: 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