polyclonal rabbit anti-hsp 60 antibody  (Accurate Chemical & Scientific Corporation)

Journal: Amino Acids

Article Title: N -Propargylglycine: a unique suicide inhibitor of proline dehydrogenase with anticancer activity and brain-enhancing mitohormesis properties

doi: 10.1007/s00726-021-03012-9

Figure Lengend Snippet: Treatment of human breast cancer cells ZR-75-1 with N -PPG, but not with T2C, activates mitochondrial PRODH degradation and increases mitochondrial expression of chaperone proteins (GRP-75, HSP-60) and the inner mitochondrial protease, YME1L1, consistent with UPR mt induction. A Confocal imaging of ZR-75-1 cells (63X oil immersion magnification) treated with vehicle (control) or N -PPG (5 mM × 24 h), PRODH (red, left panel) or GRP-75 (red, right panel), or TOM20 (green, left and right panels) stained with mouse primaries and detected by fluorochrome-conjugated secondary antibodies. Merged images confirm mitochondrial co-localization. B Western blot of mitochondrial vs. cytoplasmic expression of PRODH, GRP-75, HSP-60, tubulin, and Rieske proteins 24 h after cell culture treatment of ZR-75-1 cells with N -PPG (5 mM) or drug vehicle (C). C. Western blots comparing PRODH downregulation with 60 kDa YME1L1 upregulation in whole cell lysates of ZR-75-1 cells after 24 h and 48 h exposures to N -PPG (5 mM) or vehicle (C). Upon import into mitochondria, cleavage of the mitochondrial targeting sequence from newly induced 80 kDa cytosolic YME1L1 produces the mitochondrial membrane-localizing and longer lasting 60 kDa YME1L1 (Hartmann 2016). D Western blots of ZR-75-1 whole cell lysates comparing expression of PRODH (68 kDa), YME1L1 (60 kDa), β-actin (42 kDa), and mitochondrial Rieske (30 kDa) after 24 h cell culture treatment with vehicle (C), N -PPG (5 mM), or T2C (5 mM)

Article Snippet: Antibodies used in this study included mouse monoclonals against β-actin (C4), PRODH (A-11), TOM20, and Rieske FeS IgG (A-5) from Santa Cruz Biotechnology (Santa Cruz, CA); HRP-conjugated goat anti-mouse secondary from Bio-Rad Laboratories, Inc. (Hercules, CA); α-tubulin mouse monoclonal (T9026) from MilliporeSigma (St. Louis, MO); YME1L1, GRP-75, and HSP-60 rabbit polyclonals from ProteinTech™ (Rosemont, IL); Alexa 488 goat anti-mouse and 594 goat anti-rabbit secondaries from Life Technologies (Thermo Fisher Scientific, Carlsbad, CA).

Techniques: Expressing, Imaging, Staining, Western Blot, Cell Culture, Sequencing

Journal: Amino Acids

Article Title: N -Propargylglycine: a unique suicide inhibitor of proline dehydrogenase with anticancer activity and brain-enhancing mitohormesis properties

doi: 10.1007/s00726-021-03012-9

Figure Lengend Snippet: Oral N -PPG treatment induces expression of the mouse brain mitochondrial protease YME1L1 . A Total RNA extracted from single control (C) and 200 mg/kg N -PPG-treated mouse brains analyzed by RTqPCR to show 1.7-fold induction of chaperone HSP-60 transcripts and 1.4-fold induction of mitochondrial protease YME1L1 transcripts relative to housekeeping transcripts (GAPDH and β2 M) following 9 days of oral N -PPG treatment. B Violin plots (median: dark line, open box: Q1–Q3, distribution with max./min. values) of PRODH and YME1L1 gene expression values from RNAseq analysis of brain samples from all four control and all four 50 mg/kg N -PPG-treated mice (p values determined by Wilcoxon test). C Western blots of whole brain protein lysates from all saline-treated control and 50 mg/kg N -PPG-treated mice, as analyzed in Fig. B, with bar graphs below western blots showing image analysis quantification (mean ratio values ± SD) of 80 kDa YME1L1 expression normalized to either 42 kDa β-actin or 30 kDa Rieske, indicating a near 40% increase in mean YME1L1 expression following N -PPG treatment (ANOVA F test p values)

Article Snippet: Antibodies used in this study included mouse monoclonals against β-actin (C4), PRODH (A-11), TOM20, and Rieske FeS IgG (A-5) from Santa Cruz Biotechnology (Santa Cruz, CA); HRP-conjugated goat anti-mouse secondary from Bio-Rad Laboratories, Inc. (Hercules, CA); α-tubulin mouse monoclonal (T9026) from MilliporeSigma (St. Louis, MO); YME1L1, GRP-75, and HSP-60 rabbit polyclonals from ProteinTech™ (Rosemont, IL); Alexa 488 goat anti-mouse and 594 goat anti-rabbit secondaries from Life Technologies (Thermo Fisher Scientific, Carlsbad, CA).

Techniques: Expressing, Western Blot