Fig. 1
Evaluation of adenylate kinase 4 (AK4) expression and intracellular localization in HeLa cells. a Left panel: western blotting evaluation of AK4 (25 kDa) and alpha-tubulin (55 kDa) expression at the indicated time-points under hypoxic conditions (1 % O2). Right panel: band densities were quantified using Image Lab (BioRad) and normalized to the internal control (alpha-tubulin). *P < 0.05, **P < 0.01. b Left panel: western blotting analysis of AK4 expression after deferoxamine (DFO) treatment. Right panel: quantified data normalized by internal control alpha-tubulin were conducted by using Image Lab (BioRad). *P < 0.05, **P < 0.01. c Left panel: immunohistochemical evaluation of the intracellular localization of AK4. Upper panel: normoxia; lower: hypoxia (1 % Day 2); right panel: subcellular localization of AK4. HK2 (100 kDa) was an hypoxic marker, ATP5a (54 kDa) was a mitochondrial marker, and alpha-tubulin (55 kDa) was the loading control. d AK4 expression in a stable transformant containing AK4 tagged with GFP on its C terminus. In addition to internal AK4 (25 kDa), overexpressed AK4:GFP (50 kDa) signaling was also detected. VDAC or β-Actin is used for mitochondrial loading control or total protein loading control, respectively. e Intracellular localization of the AK4GFP stable transformant. Top: GFP, center: MitoTracker Red, bottom: merged