Journal: The Journal of Biological Chemistry
Article Title: ?1-Integrin-Collagen Interaction Suppresses FoxO3a by the Coordination of Akt and PP2A *
Figure Lengend Snippet: PTEN inhibition increases fibroblast proliferation via high Akt activity. A , upper panel , RT-PCR. Serum-starved fibroblasts were plated on collagen-coated dishes for the indicated times. The cells were lysed and total RNA was isolated by TRIzol. RT-PCR was performed as described under “Materials and Methods.” Lower panel , pulse-chase analysis. Fibroblasts were incubated in methionine-free medium followed by DMEM containing [ 35 S]methionine as outlined under “Materials and Methods.” The cells were then plated on collagen-coated dishes in DMEM + 10% fetal calf serum for the indicated times. The cells were lysed, PTEN was immunoprecipitated from labeled cell extracts and resolved by SDS-PAGE. Radiolabeled PTEN was visualized by autoradiography. B , human lung fibroblasts were infected with adenovirus expressing wild type PTEN or mutant PTEN followed by serum starvation for 1 day. Cells were then attached to collagen-coated plates (100 μg/ml) for 48 h. Total Akt, phosphorylated Akt, and FoxO3a expression levels were then measured. Wt , wild type PTEN; Mu , phosphatase-dead mutant PTEN; Vec , empty vector. C , PTEN −/− and PTEN +/+ cells were serum-starved for 24 h followed by attachment to collagen-coated plates as a function of time. FoxO3a, Ser-253-phosphorylated FoxO3a, and Ser-473-phosphorylated Akt levels were measured. Glyceraldehyde-3-phosphate dehydrogenase ( GAPDH ) was used as a loading control. D , PTEN −/− fibroblasts infected with adenovirus expressing wild type PTEN ( Wt ), phosphatase-dead mutant PTEN ( Mu ), or empty vector ( Vec ) were attached to collagen-coated plates for 60 min. FoxO3a and Ser-253-phosphorylated FoxO3a expression levels were then measured. GAPDH was used as a loading control. E , 3,000 PTEN −/− and PTEN +/+ cells were incubated in DMEM and cell numbers were counted at 72 h as described under “Materials and Methods.” The results are representative of three different experiments. F , serum-starved lung fibroblasts infected with adenovirus expressing hyperactive Akt ( Akt H ) or dominant Akt ( Akt DN , T308A, S473A) were seeded on collagen-coated plates for 30 min. FoxO3a, Ser-253 phosphorylated FoxO3a ( p-FoxO3a ), and Ser-473 phosphorylated Akt ( p-Akt ) levels were measured. GAPDH was used as a loading control.
Article Snippet: Anti-Akt and anti-phospho-Akt (Ser-473) antibodies were obtained form Cell Signaling Technologies.
Techniques: Inhibition, Activity Assay, Reverse Transcription Polymerase Chain Reaction, Isolation, Pulse Chase, Incubation, Immunoprecipitation, Labeling, SDS Page, Autoradiography, Infection, Expressing, Mutagenesis, Plasmid Preparation