Eif3i

Protein extraction and western blotting were performed as previously described[14 (link)]. Cell lysates were extracted with the T-PER tissue protein extraction reagent (Pierce, Rockford, IL) with a cocktail of proteinase inhibitors (Roche Applied Science, Switzerland) and a cocktail of phosphatase inhibitors (Roche Applied Science). Equal amounts of total proteins (20 μg) were separated by 10% SDS-PAGE and transferred onto PVDF membrane using a Bio-Rad SemiDry apparatus. After blocking for nonspecific binding, the membranes were incubated with anti-CLU (1:200 dilution; Santa Cruz Biotechnology), MMP13 (1:200 dilution; Santa Cruz Biotechnology), p-Akt (1:1000 dilution; Cell Signaling Technology), Akt (1:1000 dilution; Cell Signaling Technology), EIF3I (1:800 dilution; Abcam, Hong Kong, China) or GAPDH (1:5000 dilution; Kang-Chen, Shanghai, China) overnight at 4°C, followed by HRP-conjugated secondary antibodies for 1 h at room temperature. After washing three times in TBST, protein bands were visualized using chemiluminescence detection.

Cells were lysed by RIPA buffer supplemented with cocktail proteinase inhibitor. Then the samples were separated by SDS-PAGE gel electrophoresis, and electroblotted onto polyvinylidene fluoride membrane. After blocked with 5% BSA or nonfat milk, membranes were incubated overnight at 4°C with primary antibodies as indicated. Blots were developed with secondary horseradish peroxidase (HRP)-conjugated antibody, and signals were exposed onto x-ray films. The following antibodies were for Western blot: eIF3i(Abcam), phospho-AKT (p-AKT, Ser473; Cell Signaling technology), AKT (Cell signaling technology), phospho-ERK1/2 (p-ERK1/2, Thr202/Tyr204; Cell signaling technology), ERK1/2 (Cell signaling technology), VEGFR2 (Abcam), GAPDH (Tianjin sungene Biotech), Tubulin (Tianjin sungene Biotech).