Western blotting was performed as previously described20 (link),27 (link),54 (link),58 ,62 (link). The concentration of the lysates was determined by TaKaRa BCA Protein Assay Kit (T9300A), following the manufacture’s protocol. A3A and A3B were blotted by either a rabbit serum that reacted to both A3A and A3B20 (link). Other antibodies used in this study were as follows: rabbit anti-keratin 10 (K10) (sab4501656, Sigma), rabbit anti-GAPDH (G9545, Sigma), horseradish peroxidase (HRP)-conjugated anti-rabbit IgG (GE Healthcare, Little Chalfont, UK), mouse anti-HA (Invivogen, San Diego, California), and anti-mouse IgG–HRP (GE Healthcare).
Subcellular fractionation of W12 cells was performed using a Mitochondria Isolation Kit for Cultured Cells (Thermo Fisher, 89874) following the manufacturer’s instructions. After the removal of nuclei and unlysed cells, supernatants were spun down at 3000 × g for 15 minutes at 4 °C to harvest mitochondria with minimum contamination of lysosomes or peroxisomes. Anti-HSP-60 (GeneTex, Irvine, California, GTX110089) and β-tubulin (GeneTex, GTX101279) antibodies were used to validate the purification of the mitochondrial and cytosolic fractions, respectively.
Subcellular fractionation of W12 cells was performed using a Mitochondria Isolation Kit for Cultured Cells (Thermo Fisher, 89874) following the manufacturer’s instructions. After the removal of nuclei and unlysed cells, supernatants were spun down at 3000 × g for 15 minutes at 4 °C to harvest mitochondria with minimum contamination of lysosomes or peroxisomes. Anti-HSP-60 (GeneTex, Irvine, California, GTX110089) and β-tubulin (GeneTex, GTX101279) antibodies were used to validate the purification of the mitochondrial and cytosolic fractions, respectively.
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