Pakt308

Protein samples were prepared with RIPA buffer (Thermo Scientific Inc., Waltham, MA, USA) containing 1% protease inhibitor. Equal weight of total protein was separated by electrophoresis on SDS/PAGE. After the proteins had been transferred onto a polyvinylidene difluoride membrane (Millipore, Bedford, MA, USA), the blots were incubated with blocking buffer (1 X PBST and 5% skim milk) for 1 hour at room temperature and then hybridized with primary antibodies overnight at 4°C, followed by incubation with horseradish peroxidase-conjugated secondary antibody for 1 hour at room temperature. The blots were obtained by X-ray film exposure, and the intensities were quantified by densitometry analysis (Digital Protein DNA Imagineware, Huntington Station, NY).
The primary antibodies used in this study were anti-Flag (Sigma-Aldrich Co. LLC., St. Louis, MI, USA, F1804), AKT (cell signaling, #9272), p-Akt-308 (cell signaling, #13038), p-AKT-473 (cell signaling, #4060), Bad (cell signaling, #9239), p-Bad (cell signaling, #5284), Bax (cell signaling, #2772), cleaved-caspase-3 (cell signaling, #9661), cleaved-PARP (cell signaling, #5625), IL-6 (R&D system, #6708) and beta-actin (sigma, A5316).

For immunoblotting analysis, protein lysates were loaded onto a 10% SDS-polyacrylamide gel for electrophoresis and transferred to a PVDF membrane. Proteins were identified by incubating the membrane with primary antibodies followed by horseradish peroxidase-conjugated secondary antibodies and enhanced chemiluminescence solution (NEN Life Science, Boston, MA, USA). The primary antibodies used in this study were AKT, pAKT308, and pAKT473 from Cell Signaling Technology and ERBB2, YES1, and KDELR3 from Abcam.

HT29 cells (5 × 10⁵) were treated with deoxyshikonin in a six-well culture plate. After 48 h, we harvested the cells on ice and cleavage in RIPA lysate with 1 mM PMSF and phosphatase inhibitor for 30 min. Then, the cells were centrifuged at 12,000 rpm for 30 min at 4 °C. Next, we placed the sample on a vortex mixer, and heated in a 90 °C bath for 10 min. For tumour tissues, we placed the small pieces in lysis buffer and sonicated for 8–10 s. Then, the samples were centrifuged at 12,000 rpm for 30 min at 4 °C. After preparing the protein samples, we performed the Western blotting according to standard protocols. Briefly, the PVDF membrane containing the target protein was washed with TBST solution, incubated with 5% nonfat milk solution and primary antibodies: PI3K, p-PI3K, Akt, p-Akt308, mTOR and β-actin (Cell Signaling Technology, Boston, MA). The suspensions were gently shaken overnight at 4 °C. Then, we add HRP-conjugated polymer-tagged secondary antibodies (Abcam, Cambridge, MA) with diluted in blocking solution and incubated for 1 h on a shaker at room temperature. Lastly, the protein bands were captured using Western blotting detection system (DNR Bio-Imaging Systems, Jerusalem, Israel). Signal intensity was quantified by densitometry with the Gel-pro Analyzer (Media Cybernetics, Rockville, MD). All experiments were done in triplicate.