Expression plasmids utilized had been: pCGp19ARF, pCGc-myc, pC1-Neo-b-catenin XL S33Y , and pQmE7 . Plasmid pIRES2-EGFP expressing EGFP was implemented for handle transfections. The primary antibodies used were: anti-p53 , anti-Mdm2 , anti-Bax , antip19ARF , anti-b-catenin , anti-3F12 , anti-c-myc , anti-phos histone H2AX , and anti-actin . The secondary antibodies used in Western blot analysis have been: biotinylated sheep anti-mouse and biotinylated donkey anti-rabbit , alkaline phosphatase conjugated with streptavidin or HRP-conjugated with streptavidin . Cells and transfections. ARF_/_ MEFs have been cultured at 5% CO2 and 37 _C in IMDM supplemented with 10% fetal calf serum . Cells were transfected with 5 lg of plasmid, for co-transfections five lg of the two plasmids were used for transfection. Transfections were carried out with ExGen in vitro reagent according to the producer _s directions.
Wortmannin was additional to the culture medium two h right after transfection. Cells were incubated with wortmannin 22 or 46 h posttransfection selleck chemicals oral MEK inhibitor according to experiment. To avoid any adjustments in cellular responses caused by transient transfection, we utilized polyethylenimine transfection. This way has become reported not to have an effect on both p53 pathways or cellular responses . The suitability of this approach for transfection experiments was also confirmed by us, for the reason that we did not detect any improvements in control transfected cells compared to untransfected cells. Western blot. For protein detection, both adherent and floating cells were collected and washed twice with PBS and lysed on ice for one h in lysis buffer containing 150 mM NaCl, 50 mM Tris?HCl , 1% Triton X-100, supplemented using the total protease inhibitor mixture .
Protein concentration from the supernatant was estimated by utilizing Bio-Rad R547 reagent . Equal quantities of protein from every single lysate had been analyzed by SDS?Webpage gels and transferred to a PVDF membrane. Equivalent protein loading per lane was verified by probing the membranes with monoclonal antibody against actin. Detection of apoptotic cells by annexin-V staining. Apoptosis was detected 48 h posttransfection by staining with the annexin-VFLUOS staining kit . Each floating and adherent cells had been collected and washed with PBS. Just after washing, cells were incubated with annexin-V-fluorescein or annexin-V-phycoerythrin as indicated through the manufacturer. The cells have been analyzed using a FACSCalibur cell sorter and CellQuest Professional system .
Immunofluorescence analysis. Cells have been grown on coverslips. Twenty four hours posttransfection cells had been incubated with 20 nM mitotracker additional to culture medium for 30 min to visualize mitochondria. Cells were fixed with 4% paraformaldehyde in PBS for 15 min at room temperature and permeabilized with 0.2% Triton X-100 in PBS for ten min on ice.