Stably-transfected cells were grown in 4-chamber slides (Thermo Scientific Nunc) until 60–80% confluence. Living cell fluorescent images were captured using an AMG EVOS FL Cell Imaging System with a Plan Fluorite 40×/0.65 objective.
Immunoblots were used to confirm the redistribution of HKII and mutated HKII proteins into the cytoplasm from the mitochondria. The enriched-mitochondrial fractions and cytoplasmic fractions were collected and processed as described previously [9 (link)]. Equal concentrations of protein were separated by 10% SDS/PAGE and transferred to Immobilon-P membrane (Millipore Corp.) by electroblotting. HKII, mitochondrial Hsp70 (mtHsp70), and β-actin were identified by immunoblotting using the following antibodies purchased from Thermo Scientific; anti-HKII, anti-mtHsp70, anti-β-actin, anti-mouse-HRP, anti-rabbit-HRP. Antibodies targeting β-actin and mtHsp70 were used as loading controls for cytoplasmic and mitochondrial fractions, respectively. Protein was detected by chemi-luminescent signalling (Pierce ECL) and the immunoblots were scanned and analysed by densitometry using UN-SCAN-IT software (Silk Scientific, Inc.). The densitometry results were expressed as a ratio of the intensity of HKII to that of β-actin or Hsp70 from the same source.
Immunoblots were used to confirm the redistribution of HKII and mutated HKII proteins into the cytoplasm from the mitochondria. The enriched-mitochondrial fractions and cytoplasmic fractions were collected and processed as described previously [9 (link)]. Equal concentrations of protein were separated by 10% SDS/PAGE and transferred to Immobilon-P membrane (Millipore Corp.) by electroblotting. HKII, mitochondrial Hsp70 (mtHsp70), and β-actin were identified by immunoblotting using the following antibodies purchased from Thermo Scientific; anti-HKII, anti-mtHsp70, anti-β-actin, anti-mouse-HRP, anti-rabbit-HRP. Antibodies targeting β-actin and mtHsp70 were used as loading controls for cytoplasmic and mitochondrial fractions, respectively. Protein was detected by chemi-luminescent signalling (Pierce ECL) and the immunoblots were scanned and analysed by densitometry using UN-SCAN-IT software (Silk Scientific, Inc.). The densitometry results were expressed as a ratio of the intensity of HKII to that of β-actin or Hsp70 from the same source.
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