rabbit anti pmp70  (Boster Bio)

Journal: bioRxiv

Article Title: The ketone body β-Hydroxybutyrate mediated epigenetic chromatin β-hydroxybutyrylation protects kidneys

doi: 10.1101/2024.12.18.628574

Figure Lengend Snippet: (A) Proteomics data depicting upregulation of Pex11γ level in BHB supplemented male rat kidney. Data are mean± SEM, *p< 0.05. (B-C) Representative images (8x and 40x magnifications) of kidney sections with immunostaining for peroxisomes (Pmp70, brown color); (B) control S rats and (C) 1,3-butanediol fed rats. Relatively higher intensity of brown color was observed in the BHB group compared to control. IHC-PMP: Immunohistochemistry-peroxisomal membrane protein.

Article Snippet: After blocking with horse serum, the sections were incubated overnight with rabbit anti-PMP70 antibody (ThermoFisher Scientific, Cat# PA1-650, 1:100 dilution).

Techniques: Immunostaining, Control, Immunohistochemistry, Membrane

Journal: bioRxiv

Article Title: An inhibitor targeting glycosome membrane biogenesis kills Leishmania parasites

doi: 10.1101/2024.09.13.612636

Figure Lengend Snippet: A) Wild-type L. tarentolae promastigotes (LtP) were treated with either DMSO(-) or 12 μM of Compound 1 (+) for 24 h. Digitonin fractionation was performed to investigate the mislocalization of glycosome matrix proteins. Enolase was used as cytosolic marker. The PTS1 proteins glycerol kinase (GK) and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) served as glycosome matrix markers for PTS1 import, while aldolase served as a marker for glycosomal PTS2 import. The mitochondrial heat shock protein (mtHSP70) was used as a mitochondrial matrix marker to confirm that mitochondria were unaffected. Compared to the untreated group, GK, GAPDH, and aldolase were released from Compound 1-treated cells at lower levels of digitonin. The result indicated a partial mislocalization of glycosomal matrix proteins to cytosol upon Compound 1 treatment. B) LtP cells treated with DMSO or Compound 1 were fixed after 24 h of incubation and stained with anti- Tb Aldolase primary antibody. The red punctuate staining indicates the glycosomal localization of aldolase. Compared to the DMSO control group, cells treated with Compound 1 showed bigger red puncta, which might be related to enlarged or cluster glycosomes, suggesting a mild effect of Compound 1 on glycosomes. C) Human T-REx-293 cells were incubated with three concentrations of Compound 1 for 48 or 72 h and analyzed via immunoblotting using 3-ketoacyl-CoA thiolase (Thiolase) primary antibody. Precursor thiolase (p-Thiolase) is proteolytically processed upon peroxisomal import into its mature form (m-Thiolase), which runs at 44 kDa and serves as an indicator for peroxisomal import. Compound 1-treated wild-type (WT) cells displayed an m-Thiolase pattern similar to DMSO-treated cells, indicating that the peroxisomal protein import is normal. PEX3- and PEX19-knockout (KO) cells, in which p-Thiolase is abundant due to the absence of peroxisomes, were used as controls. D) Human T-REx293 cells, PEX3-KO, and PEX19-KO cells were grown on coverslips and treated with DMSO or 12 μM of Compound 1 for 72 h. Peroxisomes were labeled using anti-PMP70 antibody, whereas nuclei were stained with DAPI. Compound 1-treated cells showed a peroxisome pattern like that of the control (DMSO-treated) cells, indicating normal peroxisome morphology. No peroxisomes were observed in PEX3-KO and PEX19-KO cells.

Article Snippet: Afterward, cells were permeabilized using 1% Triton X-100/PBS for 5 min and blocked with 1% BSA/PBS for 1 h. Following, cells were incubated with primary antibodies rabbit anti-PMP70 (1:500, Invitrogen) for 30 min and secondary antibody goat anti-rabbit secondary antibody (1:200, Alexa Fluor™ 594) for 10 min under light protection.

Techniques: Fractionation, Marker, Incubation, Staining, Control, Western Blot, Knock-Out, Labeling

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