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Fig. 7. Deletion of the PINCH1 gene in primary MEFs. (A,B) Phase contrast pictures of untreated (A) and of Cre transduced PINCH1fl/fl (B) primary fibroblasts. Bar, 40 µm. (C) Genotyping PCR of the untreated (fl/fl) and of Cre transduced PINCH1fl/fl primary fibroblasts (–/–). A water sample (c-) served as a negative PCR control. (D) Western blot on protein extracts of untreated and of Cre transduced PINCH1fl/fl primary fibroblasts cultured in medium containing 10% FCS. (E,F) Immunofluorescent staining of untreated (E) and Cre-treated (F) PINCH1fl/fl primary fibroblasts. Cells were seeded on fibronectin-coated glass coverslips in the absence of FCS for 4 hours and stained with an affinity purified anti-PINCH1 antibody. Focal adhesions and F-actin were visualized with anti-vinculin antibody and with TRITC-conjugated phalloidin, respectively. Bar, 20 µm. (G) FACS analysis of untreated (fl/fl), Cre-treated PINCH1fl/fl primary fibroblasts (–/–) stained with Alexa Fluor 488-conjugated annexin V and propidium iodide (PI); a control population of PINCH1fl/fl primary MEFs was cultured in suspension without serum to induce apoptosis (c +). In each diagram, the lower-right sector includes early apoptotic cells that are positives for annexin V but not for PI; the upper-right sector shows the percentage of dead cells that are positive for both annexin V and PI. (H) Phosphorylation of PKB/Akt in the absence of PINCH1. Cells were starved in 0.2% FCS for 4 hours, and then stimulated for 10 minutes with 10% FCS. Phosphorylation of PKB/Akt was determined in lysates from untreated (fl/fl) and Cre-treated (–/–) primary MEFs either before (0) or after (S) serum stimulation.
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