Journal: Nature Metabolism
Article Title: Impaired oxygen-sensitive regulation of mitochondrial biogenesis within the von Hippel–Lindau syndrome
doi: 10.1038/s42255-022-00593-x
Figure Lengend Snippet: a , Fluorescence images of PC12 cells treated with 50 ng ml −1 NGF at the indicated time points. Cells were stained by MitoTracker Red to visualize mitochondria and endogenous Tuj1 (neuron-specific class III beta-tubulin) was stained in green. b , Corresponding immunoblot analysis. n = 3 biological independent experiments. c , Fluorescence images of stable polyclonal PC12 cells expressing the indicated human VHL (hu VHL ) species selected with G418 (0.5 mg ml −1 ) for 2 weeks. PC12 clones were transduced for 48 h with lentivirus encoding shRNA targeting endogenous rat VHL (sh-rat VHL ) or scramble control (shSCR) and subsequently treated with NGF for 6 d. Cells were stained by MitoTracker Red to visualize mitochondria and endogenous Tuj1 was stained in green. d , Fluorescence images of polyclonal PC12 cells transduced for 48 h with lentivirus encoding shRNA targeting endogenous rat TFAM (sh- TFAM ) or scramble control (shSCR) and subsequently treated with NGF for 6 d. Cells were stained by MitoTracker Red to visualize mitochondria and endogenous Tuj1 in green. e , Corresponding immunoblot analysis. n = 3 biological independent experiments. In a , c and d , similar results were observed more than three times.
Article Snippet: Quantification of western blots was performed by ImageJ (Supplementary Fig. ) Antibodies used were: rabbit monoclonal anti-TFAM (1:1,000 dilution; Cell Signaling Technology, 8076), rabbit polyclonal anti-PKA C-α (1:1,000 dilution; Cell Signaling Technology, 4782), rabbit monoclonal anti-PHD-2/Egln1 (1:500 dilution; Cell Signaling Technology, 4835), rabbit monoclonal anti-HIF2α (1:1,000 dilution; Cell Signaling Technology, 7096), rabbit polyclonal anti-TOM20 (1:2,000 dilution; Cell Signaling Technology, 13929), rabbit polyclonal anti-TFAM (1:1,000 dilution; Abcam, ab131607), mouse monoclonal anti-GAPDH (1:2,000 dilution; Abcam, ab8245), mouse monoclonal anti-OXPHOS (1:1,000 dilution; Abcam, ab110413), mouse monoclonal anti-MT-CO1 (1:1,000 dilution; Abcam, ab14705), rabbit polyclonal anti-MT-CO2 (1:1,000 dilution; Abcam, ab91317), rabbit polyclonal anti-MT-ND1 (1:1,000 dilution; Abcam, ab181848), rabbit polyclonal anti-MT-ATP6 (1:1,000 dilution; Abcam, ab192423), rabbit polyclonal anti-MT-CYB (1:1,000 dilution; Abcam, ab81215), rabbit polyclonal anti-LONP1 (1:1,000 dilution; Abcam, ab103809), rabbit polyclonal anti-tyrosine hydroxylase (1:1,000 dilution; Abcam, ab112), rabbit polyclonal anti-hydroxyproline (1:1,000 dilution; Abcam, ab37067, GR3215743-1 and GR3179915-1), rabbit monoclonal anti-cyclin D1 (1:1,000 dilution; Abcam, ab134175), rabbit polyclonal anti-HIF1α (1:500 dilution; Novus Biologicals, NB100-479), rabbit polyclonal anti-HIF2α (1:500 dilution; Novus Biologicals, NB100-122), mouse monoclonal anti-α-tubulin (1:2,000 dilution; Sigma-Aldrich, T5168), mouse monoclonal anti-HA (1:1,000 dilution; Sigma-Aldrich, H9658), rabbit polyclonal anti-flag (1:1,000 dilution; Sigma-Aldrich, F7425), mouse monoclonal anti-PGC1α (1:1,000 dilution; Millipore, ST1202), mouse monoclonal anti-VHL (1:500 dilution; BD Biosciences, 556347), mouse monoclonal anti-VHL (1:1,000 dilution; BD Biosciences, 564183), rabbit polyclonal anti-EGLN2 (1:500 dilution; Affinity Biosciences, DF7918), mouse monoclonal anti-TUJ1 (1:2,000 dilution, Covance, MMS-435P), mouse monoclonal anti-c-Myc (1:1,000 dilution; Thermo Fisher Scientific, 13-2500) and mouse monoclonal anti-p-Ser (16B4; 1:500 dilution; Santa Cruz Biotechnology, sc-81514).
Techniques: Fluorescence, Staining, Western Blot, Expressing, Clone Assay, shRNA, Control
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