Xenbase Image ID: 170714

Supplementary Figure 12 Localization of HSD10 in Xenopus A6 cells. A6 cells were cultivated in DMEM low glucose medium diluted with 15% water at 24°C and 5% CO2. Transient transfection of A6 cells was achieved with pCS2+_myc/xHSD10 or pT-Rex-DEST30 containing cDNA for hHSD10 wt and the mutations R130C, D86G, Q165H, respectively, using lipofectamine (Invitrogen) according to the manufacturer’s instructions. (A-E) Transfected cells were cultured for 48 h, fixed on coverslips with 3.7% formaldehyde and permeabilized with 0.5% Triton X-100 and 3.7% formaldehyde in PBS for 5 min. Unspecific binding sites were blocked with 20% newborn calf serum in IIF buffer (2% newborn calf serum, 2.5% cold water fish gelatine, 10% glycerol, 0.1% Tween-20, 2% normal serum according to the second antibody in PBS). Overexpressed proteins were detected using 9E10 anti-myc antibody and mouse monoclonal anti-HSD10 antibody (Abcam). After incubation with secondary antibody cells were washed and transferred to 200 nM Mitotracker Red 580 (Invitrogen) for 30 min. The cells were then fixed in 3.7% formaldehyde, mounted in Mowiol and observed by fluorescent microscopy (Olympus SZX12). Fluorescent signal of HSD10 is seen as puncta within the cytosol. Visualization of mitochondria using Mitotracker Red 580. Merged images show clear co-localization between Mitotracker Red and HSD10 in A6 cells regardless of the protein being human (wt or mutated) or Xenopus. The anti-HSD10 antibody does not recognize the endogenous Xenopus HSD10 protein but the transfected human proteins. All proteins (wt and mutations) are stably expressed and show a similar cellular distribution pattern. Image published in: Rauschenberger K et al. (2010) Image downloaded from an Open Access article in PubMed Central. Copyright © 2010 EMBO Molecular Medicine Larger Image Printer Friendly View

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