anti heat shock protein hsp 60  (Novus Biologicals)

Journal: Experimental Physiology

Article Title: Effect of prolonged voluntary wheel running on oxidative stress and defence mechanisms in cortex and hippocampus of healthy female rats

doi: 10.1113/ep092815

Figure Lengend Snippet: FIGURE 6 Mitochondrial BER. (a) Representative immunoblots of target proteins in mitochondrial extracts from cortex and hippocampus. Proteins probed for are shown on the left. APE1, apurinic/apyrimidinic endonuclease 1; HSP60, heat shock protein 60. (b) Representative gel from in vitro AP endonuclease activity assay with mitochondrial extracts from hippocampus. S, substrate; P, product; AP, oligonucleotide with AP site; C, control oligonucleotide without a lesion. (c–f) All values are normalized to HSP60 (mitochondrial loading control) and set relative to the sedentary group mean. Bars represent means ± SD. Differences were evaluated by Student’s unpaired t-test (c) or Mann–Whitney U-test (d–f). (c) n = 10 and 8 for sedentary and running group, respectively. (d) n = 10 and 7 for sedentary and running group, respectively. (e) n = 9 and 8 for sedentary and running group, respectively. (f) n = 7 and 9 for sedentary and running group, respectively. One outlier was excluded from the analysis in the sedentary group for (e, f). Other discrepancies in sample size are due to technical issues in assays or insufficient yield from mitochondrial purification. BER, base excision repair.

Article Snippet: anti-8-oxoguanine DNA glycosylase 1 (OGG1) (Novus Biologicals, cat. no. NB100-106, 1:500, RRID: AB_10104097), mouse anti-superoxide dismutase 1 (SOD1) (Santa Cruz Biotechnology, Dallas, TX, USA, cat. no. sc-101523, 1:500, RRID: AB_2191632), mouse anti-superoxide dismutase 2 (SOD2) (Santa Cruz Biotechnology, cat. no. sc-137254, 1:200, RRID: AB_2191808), mouse anti-catalase (CAT) (Santa Cruz Biotechnology, cat. no. sc-271803, 1:500, RRID: AB_10708550), mouse anti-heat shock protein 60 (HSP60) (Santa Cruz Biotechnology, cat. no. sc-271215, 1:1000, RRID: AB_10607973), and mouse antiβ-actin (Sigma-Aldrich, St Louis, MO, USA, cat. no. A2228, 1:10,000, RRID: 476697).

Techniques: Western Blot, In Vitro, Activity Assay, Control, MANN-WHITNEY, Purification

Journal: Biology

Article Title: Nanovesicular Mediation of the Gut–Brain Axis by Probiotics: Insights into Irritable Bowel Syndrome

doi: 10.3390/biology13050296

Figure Lengend Snippet: Details of sources and concentrations of antibodies used in this study.

Article Snippet: Heat Shock Protein 60 (Hsp60) , Sc-59567 Mouse Monoclonal Santa Cruz Biotechnology, Dallas, TX, USA , 1:1000 , 1:50.

Techniques: Western Blot, Immunofluorescence

Journal: Biology

Article Title: Nanovesicular Mediation of the Gut–Brain Axis by Probiotics: Insights into Irritable Bowel Syndrome

doi: 10.3390/biology13050296

Figure Lengend Snippet: Anti-stress effect of probiotics against oxidative stress induced by H 2 O 2 in HT29. Representative images captured for Hsp60 (green) ( a ) and tight junction protein (TJP) (green) ( b ) detection using immunofluorescence. Nuclei were counterstained with DAPI (blue). The observation using confocal microscopy confirmed that the probiotic treatment reduced stress induced by H 2 O 2 in HT29 and restored the epithelial barrier. Scale bars = 50 μm. ut: untreated cells; mix 24 h: cells treated with a probiotics mix for 24 h; mix 2 h + H 2 O 2 22 h: cells pretreated with a probiotics mix for 2 h and then with H 2 O 2 for 22 h; H 2 O 2 24 h: cells treated with H 2 O 2 for 24 h; H 2 O 2 2 h + mix 22 h: cells pretreated with H 2 O 2 for 2 h and then with a probiotics mix for 22 h; mix + H 2 O 2 24 h: cells treated simultaneously with a probiotics mix and H 2 O 2 for 24 h.

Article Snippet: Heat Shock Protein 60 (Hsp60) , Sc-59567 Mouse Monoclonal Santa Cruz Biotechnology, Dallas, TX, USA , 1:1000 , 1:50.

Techniques: Probiotics, Immunofluorescence, Confocal Microscopy

Journal: Biology

Article Title: Nanovesicular Mediation of the Gut–Brain Axis by Probiotics: Insights into Irritable Bowel Syndrome

doi: 10.3390/biology13050296

Figure Lengend Snippet: Working hypothesis. Summarizing our working hypothesis, we assume that treatment with the probiotics mix induces an increase in the metabolism of the aminoacidic tryptophan as a precursor for the synthesis of kynurenine. The latter is a product metabolized by the enzyme TDO2. Kynurenine binds AhR, which acts as a ligand-dependent transcription factor capable of controlling complex transcriptional processes. AhR is a cytoplasmic transcription factor that is normally found in an inactive form bound to the co-chaperone Hsp90 and can be activated by various indole derivatives, including kynurenine. Upon binding, the Hsp90 chaperone dissociates and AhR heads into the cell nucleus, acting in favor of the host homeostasis. The probiotics mix acts on cell stress, inducing a reduction of Hsp60 level in H 2 O 2 -exposed cells, and has a positive effect on intestinal permeability, increasing the expression of junctional proteins. Furthermore, following the complex two-way communication system that characterizes the gut–microbiota–brain axis, the increased tryptophan metabolism at the intestinal level induced by probiotic intake could lead not only to increased synthesis of kynurenine but also to increased concentration of serotonin by increasing the levels of 5-HT 2C and the systemic levels. This can result in a mechanism that can be able to limit stress-related psychiatric disorders. Finally, since plasma-derived EVs and vesicles isolated from HT-29 cells showed an increase in TDO 2 protein levels following probiotic treatment; our data suggest that EVs may extend the regulation of host homeostasis by inducing protection even at the level of the central nervous system against neurodegenerative and neuropsychiatric disorders, including depression.

Article Snippet: Heat Shock Protein 60 (Hsp60) , Sc-59567 Mouse Monoclonal Santa Cruz Biotechnology, Dallas, TX, USA , 1:1000 , 1:50.

Techniques: Probiotics, Binding Assay, Permeability, Expressing, Concentration Assay, Clinical Proteomics, Derivative Assay, Isolation

Journal: Oncology Letters

Article Title: In vitro anticancer effect of azithromycin targeting hypoxic lung cancer cells via the inhibition of mitophagy

doi: 10.3892/ol.2023.14146

Figure Lengend Snippet: Effect of AZM on the autophagy and mitophagy flux under normoxic or hypoxic conditions. A549 cells were subjected to either normoxia (20% O 2 ) or hypoxia (0.3% O 2 ) for (B-D) 24 or (A) 48 h in the presence or absence of AZM (A, 10 µM; B-D, 25 µM). (A) Western blot analysis of the autophagy substrate p62, the autophagosomal marker LC3B, and the mitochondrial abundance markers HSP 60 and UQCRC1. Band intensities were normalized to β-actin expression and data were summarized in the graphs on the right. Data are presented as the mean and standard deviation (n=3). †P<0.05 and ††P<0.01 (one-way ANOVA). *P<0.05 and **P<0.01 (Tukey's test). (B) Representative images for the detection of the autophagy flux by staining with DAP ® Red (red) and DAL ® Green (green). Scale bar, 20 µm. (C) Representative images for the detection of the mitophagy flux by staining with Mtphagy ® Dye (red) and Lyso ® Dye (green). Scale bar, 20 µm. (D) Representative images for the detection of lysosomal acidification by staining with pHLys ® Red. Scale bar, 20 µm. AZM, azithromycin; Con, control; HSP 60, heat shock protein 60; UQCRC1, ubiquinol-cytochrome b-c1 complex subunit 1.

Article Snippet: The primary antibodies used in this study were rabbit polyclonal anti-poly (ADP-ribose) polymerase 1 (1:1,000; sc-7150, Santa Cruz Biotechnology), rabbit polyclonal anti-caspase-3/p17/p19 (1:1,000; 19677-1-AP, Proteintech Group, Inc., Rosemont, IL, USA), rabbit polyclonal anti-ubiquinol-cytochrome b-c1 complex subunit 1 (UQCRC1; 1:1,000; 21705-1-AP, Proteintech), rabbit polyclonal anti-p62 (1:1,000; 18420-1-AP, Proteintech, Rosemont, IL, USA), mouse monoclonal anti-heat shock protein 60 (HSP 60; 1:1,000; SPA-807, Stressgen Biotechnologies, San Diego, CA, USA), rabbit polyclonal anti-microtubule-associated protein 1 light chain 3B (LC3B; 1:1,000; NB600-1384, Novus Biologicals, Inc., Littleton, CO, USA), rabbit monoclonal anti-Bcl-2/E1B-19kDa interacting protein 3 (BNIP3; 1:1,000; #44060, Cell Signaling Technology, Danvers, MA, USA), rabbit monoclonal anti-Bcl-2/E1B-19kDa interacting protein 3-like (BNIP3L/Nix; 1:1,000; #12396, Cell Signaling Technology), and mouse monoclonal anti-β-actin conjugated with horseradish peroxidase (1:6,000; #017-24573, Fujifilm Wako Chemicals, Osaka, Japan).

Techniques: Western Blot, Marker, Expressing, Standard Deviation, Staining