Rabbit anti phosphor mtor

The antibodies used in this study include the following: rabbit anti-phosphor-mTOR, rabbit anti-mTOR, mouse anti-Phospho-p70 S6 Kinase, rabbit anti-p70 S6 kinase, rabbit anti-CD44, rabbit anti-cleaved PARP and rabbit anti-cleaved caspase-3 were all obtained from Cell Signaling Technology (Danvers, MA, USA). Mouse-anti-p32 (gC1qR) antibody was obtained from Abcam (Cambridge, MA, USA). Rabbit anti-Akt and mouse anti-Phospho-Akt antibodies were purchased from Santa Cruz Biotechnology (Dallas, TX, USA). Goat anti-mouse and anti-rabbit IgG-horseradish peroxidase-conjugates were from Pierce (Rockford, IL, USA). Mouse monoclonal anti-actin was a gift from Dr. José Manuel Hernandez-Hernández (CINVESTAV-IPN). Temsirolimus (CCI-779) and 5-fluorouracil (5-FU) were purchased from Sigma-Aldrich (St. Louis, MO, USA).

The whole-cell lysates were harvested in the RIPA buffer supplemented with protease and phosphatase inhibitors (Thermo Scientific). The protein concentration was determined using a BCA assay (Pierce, Rockford, IL, USA) according to the manufacturer’s instructions. The cell lysates were separated using 10% SDS-PAGE gels, and the proteins were then transferred to polyvinylidene fluoride (PVDF) membranes (Millipore, Billerica, MA, USA). The membranes were blocked with 5% non-fat milk in PBST buffer for 1 h at room temperature and then incubated with the indicated primary antibodies overnight at 4 °C. After washing with PBST, the membranes were incubated with goat anti-rabbit or anti-mouse secondary antibodies (1:5000, Thermo Scientific) for 2 h at room temperature. The enhanced chemiluminescence (ECL, Pierce) was then applied, and the protein bands were imaged using a ChemiDoc Imaging System (Bio-Rad), which was quantified using the Image Lab software v6.0 (Bio-Rad). The primary antibodies used in this study included rabbit anti-ELK1 (1:1000, Abcam), rabbit anti-total MTOR (1:1000, Cell Signaling, Danvers, MA, USA), rabbit anti-phosphor-MTOR (1:1000, Cell Signaling), rabbit anti-total S6K1 (1:1000, Cell Signaling), rabbit anti-phosphor-S6K1 (1:1000, Cell Signaling), and mouse anti-β-actin (1:1000, Santa Cruz Biotech, Santa Cruz, CA, USA).