mouse monoclonal heat shock protein 60  (Cell Signaling Technology Inc)

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

Journal: PLoS ONE

Article Title: Celiac Anti-Type 2 Transglutaminase Antibodies Induce Phosphoproteome Modification in Intestinal Epithelial Caco-2 Cells

doi: 10.1371/journal.pone.0084403

Figure Lengend Snippet: MS analysis and database searching of the 13 spots with differential phosphorylation level in samples from anti-TG2 treated cells and from control-IgG treated cells.

Article Snippet: Then, nonspecific binding sites were blocked in PBS containing 5% non-fat dry milk, and membranes were incubated overnight with specific mouse monoclonal antibodies recognising elongation factor 1-γ (EF1γ) (clone X5-P), translationally-controlled tumor protein (TCTP) (clone B3), 60 kDa heat shock protein (HSP60) (clone 24) (Santa Cruz Biotechnology Inc.), at a dilution of 1∶1000 in PBS containing 1% non-fat dry milk.

Techniques: Phospho-proteomics, Control, Variant Assay

Journal: PLoS ONE

Article Title: Celiac Anti-Type 2 Transglutaminase Antibodies Induce Phosphoproteome Modification in Intestinal Epithelial Caco-2 Cells

doi: 10.1371/journal.pone.0084403

Figure Lengend Snippet: Total protein extracts (150 µg) from Caco-2 cells treated with anti-TG2 antibodies or with control IgG were separated by 2-DE using linear IPG strips, pH 5.5–6.7 for EF1γ ( A ), pH 4.7–5.9 for HSP60 ( B ), pH 3.9–5.1 for TCTP ( C ). Specific proteins were identified by immunoblot analysis.

Article Snippet: Then, nonspecific binding sites were blocked in PBS containing 5% non-fat dry milk, and membranes were incubated overnight with specific mouse monoclonal antibodies recognising elongation factor 1-γ (EF1γ) (clone X5-P), translationally-controlled tumor protein (TCTP) (clone B3), 60 kDa heat shock protein (HSP60) (clone 24) (Santa Cruz Biotechnology Inc.), at a dilution of 1∶1000 in PBS containing 1% non-fat dry milk.

Techniques: Control, Western Blot