Anti akt1 sc 5298

We evaluated the effects of miRNA-548x and miR-4698 on the expression of AKT1 protein level in GBM cell lines A-172 and U251. Seventy-tow hours after transduction, the protein was extracted from cells by using RIPA lysis buffer including the protease inhibitor cocktail (Merck, Darmstadt, Germany) and concentration was evaluated using the BCA assay (Thermo Fisher Scientific, Waltham, USA). Next, equal concentrations of each specimen were separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to the polyvinylidene difluoride (PVDF) membrane. Following blocking with 5% skim milk (Merck, Darmstadt, Germany), the membrane was stored at 4 °C for overnight in the presence of primary antibodies anti-AKT1 sc-5298 (1:200, Santa Cruz Biotechnology Inc., Dallas, USA) or anti-B-actin (1:1000, Abcam, Cambridge, Britain) as the internal reference. Subsequently, the membrane was incubated with secondary antibodies conjugated with horseradish peroxidase (1:1000, Abcam, Cambridge, Britain). Lastly, for imaging, the ECL Western Blotting Substrate (Thermo Fisher Scientific, Waltham, USA) was used. The relative density of the band was analysis by Image J software version 1.52a^{17 (link)}.

A total of 300,000 cells/well were seeded into 6-well plates. After 24 h, the cells were incubated with serum- and steroid-deficient medium and treated with 1 µg/mL tetracycline for 48 h. Then, the cells were treated with solvent (0.1% DMSO (v/v) and 0.1% ethanol (v/v)) as a control or 10 µM Api alone or in combination with 1 nM E2 for 24 h. After the treatment, the cells were directly lysed in 2X Laemmli buffer. The protein extracts were denatured for 10 min at 95 °C, separated on 7.5% SDS polyacrylamide gels, and transferred to polyvinylidene difluoride membranes (Millipore). The proteins were then probed with specific antibodies. The following antibodies were used: anti-Akt1 (sc-5298, Santa Cruz Biotechnology, Dallas, TX, USA) (dilution 1:1000), anti-FOXM1 (D12D5, Cell Signaling Technology, Danvers, MA, USA) (dilution 1:1000), anti-FOXO3a (ab53287, Cambridge, MA, USA) (dilution 1:1000), anti-cyclin B1 (sc-245, Santa Cruz Biotechnology, Dallas, TX, USA) (dilution 1:1000), anti-FOXO1 (C29H4, Cell Signaling Technology, Danvers, MA, USA) (dilution 1:1000) and anti-β-actin (A1978, Sigma-Aldrich, St Louis, MO, USA) (dilution 1:5000), which served as a control for the total amount of protein. An enhanced chemiluminescence system (Immune-Star, Bio-Rad, Hercules, CA, USA) was used to detect the immunocomplexes.