Emerling et al. 10.1073/pnas.0706790105.
Fig. 7. Immunoblot analysis of PTEN. Pten-/- cells were for infected with an adenovirus encoding PTEN for 3 h in serum/antibiotic-free media and subsequently incubated for 16 h in serum-free media before harvesting. Cell lysates were analyzed by immunoblotting with an anti-PTEN antibody. The same blot was stripped and reprobed with an anti-a-tubulin antibody to control for loading.
Fig. 8. Localization of PTEN. To examine subcellular localization of PTEN mutants, Pten-/- cells were transfected with PTEN expression constructs and visualized by immunofluorescence.
Fig. 9. Expression and localization of FOXO3a adenoviruses. (A) Pten-/- cells were infected with the HA-tagged adenoviruses expressing the wild-type FOXO3a or the constitutively active FOXO3a (AAA) (100 pfu/ml) for 3 h in serum/antibiotic-free media and subsequently incubated for 16 h in serum-free media before harvesting. Cell lysates were analyzed by immunoblotting with an anti-HA antibody. The same blot was stripped and reprobed with an anti-a-tubulin antibody to control for loading. A representative blot is shown of four independent experiments. (B) To examine subcellular localization of the FOXO3a adenoviruses, Pten-/- cells were infected as in A and visualized by immunofluorescence. Immunostaining was performed with a MAb to FOXO3a, followed by immunofluorescence microscopy, as described in SI Methods.
Fig. 10. Immunoprecipitation of HIF-1a protein under normoxia, hypoxia, or DMOG. U251 cells were exposed to 21.0% O2, 1.5% O2, or DMOG (1 mM) for 16 h, and subsequently nuclear fractions were collected. Immunoprecipitation was carried out on the U251 nuclear lysates using anti-HIF-1a or an IgG control antibody followed by immunoblotting for HIF-1a.
SI Methods
Immunofluorescence Microscopy. Pten+/- or Pten-/- MEFs were cultured on glass coverslips. Cells were transfected with PTEN constructs as indicated, infected with the adenoviruses, AdFOXO3a (WT) and AdFOXO3a (AAA), or subjected to conditions as indicated. Cells were washed three times with PBS and fixed with 3.7% paraformaldehyde (PFA) for 20 min at room temperature. PFA was quenched with PBS + glycine (10 mM) for 10 min at room temperature, washed one more time with PBS, and then permeabilized/blocked using 0.3% Triton X-100 + 0.3% BSA + normal goat serum (NGS) in PBS for 30 min at room temperature. First antibody incubation was carried out with either anti-FKHRL-1/FOXO3a (Upstate Biotechnology) or anti-PTEN (BD PharMingen) for 1 h at room temperature, followed by a secondary antibody incubation with either an Alexa-Fluor 488 goat anti-rabbit antibody (Molecular Probes) or a fluorescein goat anti-mouse antibody (Molecular Probes). The coverslips then were washed three times in PBS/BSA/TX-100, removed, and mounted with Vectashield mounting medium containing DAPI (Vector Laboratories). Cells were observed under the Axioplan 2 fluorescence microscope (Carl Ziess MicroImaging, Inc.; ´63 objective) and imaged with AxioVision LE software (Carl Ziess MicroImaging, Inc.).