Supplemental Materials and Methods
Densitometric analysis of HIF immunoblots
HIF-1α and HIF-2α experimental immunoblots, along with corresponding actin and
eEF2 loading control immunoblots, were obtained on whole-cell protein extracts from
untreated or cobalt chloride-treated ES cells as described in primary Materials and
Methods section. Densitometry was performed on scanned immunoblot images using
the ImageJ gel analysis tool (Abramoff et al., 2004). The gel analysis tool was used to
obtain the absolute intensity (AI) for each experimental HIF band and corresponding
control band. Relative intensity (RI) for each experimental band was calculated by
normalizing the experimental AI to the corresponding control AI.
Immunoblot analysis of HIF stabilization in teratomas
Extracts were prepared from fresh teratomas harvested at six weeks post-injection
using NET lysis buffer (20mM Tris, 100mM NaCl, 1mM EDTA, 1% NP-40) and
quantitated by Bradford assay. Immunoblot primary antibodies used were: HIF-1α
(Mansfield et al., 2005), HIF-2α (NB100-122, Novus Biologicals), and actin (Sigma, St.
Louis, MO, USA).
References
Abramoff MD, Magelhaes PJ and Ram SJ. (2004). Biophotonics International, 11, 3642.
Mansfield KD, Guzy RD, Pan Y, Young RM, Cash TP, Schumacker PT and Simon MC.
(2005). Cell Metab, 1, 393-9.
Titles and Legends to Supplemental Figures
Supplemental Figure 1. Densitometry analysis for ES cell HIF-1α and HIF-2α
immunoblots. Experimental band intensity for each sample was determined relative to
the corresponding loading control band using the ImageJ gel analysis tool. Co,
treatment with Cobalt Chloride.
Supplemental Figure 2. Homozygous Type 2B teratomas display sub-maximal HIF
stabilization compared to null. Immunoblots for HIF-1α (A), and HIF-2α (B), and eEF2
and actin (loading controls, respectively) on extracts prepared from fresh teratoma
tissue harvested at six weeks post-injection.