Enhanced chemical luminescence

The effect of miR-34a transfection on the expression of proteins associated with osteoblast-related factors was evaluated by western blotting, in which protein levels were normalized against those of GAPDH. Seventy-two hours after transfection with the N-Ac-l-Leu-PEI/miR-34a nanocomplex (4 wt/wt ratio, 2 μg miR-34a), MG63 cells were harvested, washed twice with ice-cold PBS and lysed with RIPA lysis buffer on ice for 5 min. The lysates were centrifuged at 14 000 × g for 15 min and supernatants collected. Protein concentrations were determined using a BCA protein assay kit. Equal amounts of protein were subjected to SDS-PAGE and subsequently transferred to PVDF membranes by electroblotting. The membranes were blocked in PBS containing 5% skimmed milk and 0.1% Tween-20 for 1 h, and then incubated with desired antibodies at 4 °C overnight. Membranes were subsequently incubated with an HRP-goat anti-rabbit IgG (H + L) antibody for 1 h and detected using enhanced chemical luminescence (Amersham, UK).

Cells were lysed using RIPA buffer for 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 3 (PFKFB3) detection. To analyze nuclear factor erythroid 2-like 2 (Nrf2) levels, cytosolic and nuclear compartments were extracted first with cytosol extraction buffer containing HEPES (10 mM) pH 7.9, KCl (10 mM), EDTA (0.1 mM), 0.3% NP-40, and protease inhibitors and then with nucleus extraction buffer containing HEPES (20 mM) pH 7.9, NaCl (0.4 mM), EDTA (1 mM), 0.25% glycerol, and protease inhibitors. The cell lysate was subjected to Western blot analysis using anti-PFKFB3 antibody (Ab) (Abgent, San Diego, CA, USA), anti-Nrf2 Ab (Santa Cruz Biotechnology, Dallas, TX, USA), anti-actin Ab (Bethyl Laboratories Inc., Montgomery, TX, USA), or anti-lamin A/C Ab (Santa Cruz) as a primary Ab and horseradish peroxidase-conjugated anti-rabbit IgG Ab (Thermo Fisher Scientific) as a secondary Ab. Signals were detected using enhanced chemical luminescence (Amersham, Piscataway, NJ, USA). The immunoblot was digitized using an office scanner (UMAX Astra 4100, Taipei, Taiwan), and the intensity of the band of the expected molecular size was quantitated using Image Gauge V 4.0 (Fujifilm Corporation, Tokyo, Japan). The band intensity of PFKFB3 and cytosolic Nrf2 was normalized to actin, and nuclear Nrf2 was normalized to lamin.