Phosphorylation of eIF2α mediates the antiproliferative and proapoptotic functions of PTEN. (A) eIF2αS/S and eIF2αA/A MEFs were transfected with pcDNA-GFP-zeocinR vector (GFP), as well as pcDNA vector encoding either FLAG-PTEN WT cDNA or FLAG-PTEN C124S cDNA. Forty eight hours after transfection, cells were visualized by microscopy. (B) Protein extracts (50 μg) from eIF2αS/S and eIF2αA/A MEFs treated as described in (A) were analyzed by Western blotting with antibodies against caspase-3, the FLAG epitope, Bcl-xL, Bax, or actin. (C) eIF2αS/S and eIF2αA/A MEFs were transfected as described for (A) and protein extracts (50 μg) were analyzed for the activity of caspase 3 with Ac-VEID-AFC as substrate. The assay was performed in triplicate; error bars indicate SD, n = 3. The group difference was tested by ANOVA (P D) eIF2αS/S and eIF2αA/A MEFs were infected with either empty pBABE retroviruses (as a control) or pBABE retroviruses containing either PTEN WT or PTEN C124S cDNAs. After selection in puromycin (2.5 μg/ml) for 3 weeks, colonies were fixed and stained with crystal violet. Plates were scanned, and quantification of the clones on the images was performed with Scion Image 4.0.3.2 software. Histograms show quantification of results from three independent experiments; error bars indicate SD, n = 3. The group difference was tested by ANOVA (P
Scion Image Download 4.0.3.2 32
The study was carried out on 40 human fetuses (20 females and 20 males) aged from 15 to 29 weeks (total body length v-pl from 130 to 345 mm). Following methods were used: anthropological, preparatory, image acquisition with a digital camera, computer measurement system Scion for Windows 4.0.3.2 Alpha and Image J (accuracy up to 0.01 mm without damaging the unique fetal material) and statistical methods. 2ff7e9595c
Comments