Alpha actinin

After homogenizing the samples with RIPA lysis buffer containing protease inhibitors, protein concentrations were detected with the Bradford assay. Before electrophoresis on an SDS-agarose gel, an equal amount of cell lysates was added to protein loading dye and boiled for 5–10 min. The separated proteins were blotted electrophoretically onto a polyvinylidene difluoride membrane (Millipore Corp., Bedford, MA, USA). Nonspecific binding was blocked with 5% nonfat milk in Tris-buffered saline for 1 hour at room temperature. The following primary antibodies were then applied overnight at 4° C: alpha-actinin (Santa Cruz Biotechnology, Santa Cruz, CA, USA), CD164 (R&D Systems, Inc. Minneapolis, USA), CXCR4, Akt/p-Akt, mTOR/p-mTOR, OCT4, caspase-3/cleaved caspase-3, caspase-9/cleaved caspase-9, Beclin-1, p62, LC3B (Cell Signaling, Massachusetts, USA), mTORC1 (Proteintech Group, Inc, Rosemont, USA). After washing, HRP-conjugated secondary antibodies diluted in blocking buffer were applied for 1h at room temperature. Immune complexes were detected by enhanced chemiluminescence, and images captured on X-ray film.

Cell lysates were harvested in lysis buffer (100 mM Tris–HCl pH 8.0, 150 mM NaCl, 0.1% SDS, and 1% Triton 100) at 4°C and were separated by SDS-PAGE electrophoresis. The proteins were then transferred to a polyvinylidine difluoride membrane (Millipore, USA) and were detected using antibodies against alpha-actinin (ACTN), p53, p21, cyclin D1, Twist, MMP-2 (Santa Cruz Biotechnology, USA), ERK, p-ERK, Akt, p-Akt, AMPKα, p-AMPKα, ACC, p-ACC, Snail (Cell signaling, USA), GFAP (BD Pharmingen, USA), vimentin (GeneTex Inc.), and ME2 (Sigma, USA).