West dura extended chemiluminescent detection

Cells were lysed in Staph-A buffer (1.6 mM NaH₂PO₄; 8.6 mM Na₂HPO₄; 1% Triton X-100; 0.1% SDS; 0.1% NaCl; 0.5% NaDoc; 2 mM AEBSF; 20 mg/mL each of aprotinin and leupeptin) and the obtained protein lysates (100 µg each) were subjected to SDS-PAGE electrophoresis and transferred to PVDF membrane (Millipore, Burlington, MA, USA). Blots were probed with the following monoclonal antibodies: anti-PARP (Abcam, Cambridge, MA, UK), anti-phospho-Histone γH2AX (Millipore), anti-TPX2 (Santa Cruz Biotechnology, Dallas, TX, USA), and anti-c-MYC (Santa Cruz Biotechnology); and polyclonal antibodies: anti-GOLPH3 (Abcam), anti-Beclin-1 (Novus Biologicals, Littleton, CO, USA), anti-LC3A (Cell Signaling Technology Inc., Danvers, MA, USA), anti-MYO18A (Abnova, Taipei, Taiwan), anti-Histone H2AX (Sigma, St Louis, MO, USA), and anti-MYCN (Novus Biologicals). Proteins were visualized by West Dura Extended chemiluminescent detection (Thermo Scientific, Carlsbad, CA, USA) using HRP-conjugated secondary antibodies (Thermo Scientific). Blots were re-probed with anti-β-actin (Santa Cruz Biotechnology) as loading control. Bands signal intensity was measured by densitometry using Image Lab 6.0 software (ChemiDoc, Bio-Rad, Hercules, CA, USA), and normalized to the loading control.

Appropriate amounts of microsomal proteins (100 μg), obtained from control and LS-G6pc^−/− livers, were subjected to 10% SDS-PAGE electrophoresis, transferred to PVDF (Millipore, Billerica, MA, USA) and probed with a polyclonal anti-G6Pase-α antibody (Santa Cruz Biotechnology, Dallas, TX, USA). Immunocomplexes were visualized by West Dura extended chemiluminescent detection (Thermo Scientific, Walthman, MA, USA) using a HRP-conjugated secondary antibody (Pierce, Rockford, IL, USA,).

Human NB cells, grown in normoxic or hypoxic conditions, were lysed in a buffer containing 1,6 mM NaH₂PO₄, 8,6 mM Na₂HPO₄, 1% Triton X-100, 0,1% SDS, 0,1% NaN₃, 0,1M NaCl, 0,5% NaDoc, 2 mM AEBSF, and 20 mg/ml each of aprotinin and leupeptin. Lysates (100 μg each) were subjected to 8% SDS-PAGE electrophoresis, transferred to PVDF membrane (Millipore, Billerica, MA, USA), and probed with anti-VEGFA (1:100), anti-PHD3 (1:1000), anti-PDK1 (1:500), anti-PFKFB4 (1:200), anti-HIF-1α (1:250), and anti-N-Myc (1:3000) antibodies. Proteins were visualized by West Dura extended chemiluminescent detection (Thermo Scientific) using HRP-conjugated secondary antibodies against mouse or rabbit (Pierce, Rockford, IL, USA). As loading control, blots were reprobed with mouse monoclonal anti-β-actin antibody (sc-47778 by Santa Cruz Biotechnology) (1:200). PBS and lysis-buffer were allowed to equilibrate in the hypoxic incubator for 1 hr before using them to wash and collect cells cultured in hypoxic conditions. (https://dx.doi.org/10.17504/protocols.io.jtvcnn6)