Phospho-Akt Stimulation and Western Blotting

4T1 cells were starved in DMEM/F12 supplemented with 0.5% FCS for 24 h, then washed and resuspended in serum free DMEM/F12 at 4 × 10⁷ cells/ml. After 5 min pre-incubation at 37°C, the cells were stimulated with equal volume of vASC or ASC.B6 conditioned media for different time points. The stimulation was stopped by adding 2× lysis buffer [1 × lysis buffer contains: 50 mM TrisHCl pH 7.4, 150 mM NaCl, 2 mM EDTA, 20 mM NaF, 200 μM Na₃VO₄, 1 mM PMSF, cOmplete™ Mini EDTA-free Protease Inhibitor Cocktail (Roche)]. After 30 min lysis on ice the samples were centrifuged for 15 min at 13,000 × g and then the lysates were boiled with 2× sample loading buffer for 5 min. Cells lysates from 1.5 × 10⁵ cells were run on a 10% SDS-PAGE, and transferred to PVDF membranes. The membranes were blocked with 3% gelatin from cold-water fish skin in PBS for 1 h at room temperature, and then incubated with anti-phospho Akt (Ser473) antibody (Cell Signaling Technology, #9271) overnight at 4°C. After washing and incubating with swine anti-rabbit Ig-HRP (DAKO) for 1 h at room temperature, the immunoreactive proteins were visualized using WesternBright ECL HRP substrate (Advansta), and the chemiluminescence signal was detected with Odyssey Imaging System (LI-COR Biotechnology). To re-probe with different antibodies, the membranes were stripped in stripping buffer (Re-Blot Plus Strong, Millipore) for 15 min at room temperature. The amount of loaded proteins was tested with anti-Akt (Pan) antibody (Cell Signaling Technology, #2920), followed by anti-mouse Ig-HRP (DAKO).