Whole-cell Lysate Preparation and Protein Analysis

Whole-cell lysates were prepared using RIPA buffer as described previously [39 (link)] and analyzed using the following primary antibodies: anti-β-actin, anti-Sp1 (PEP2), anti-cyclin E, anti-ZEB1, and anti-GAPDH (Santa Cruz Biotechnology, Santa Cruz, CA, USA); anti-myc (Upstate Biotechnology, Lake Placid, NY, USA); anti-integrin α5 and anti-Tie2 (BD Biosciences, San Jose, CA, USA); anti-vimentin (Sigma, St Louis, MO, USA); anti-phospho-c-Jun N-terminal kinase (JNK) (T183/Y185), anti-phospho-extracellular signal-regulated kinase 1/2 (ERK1/2), anti-ERK1/2, anti-phospho-Akt (S473), anti-Akt, anti-survivin, anti-cyclin D1, anti-JNK, anti-phospho-VEGFR2 (Y996), anti-phospho-VEGFR2 (Y1175), anti-VEGFR2, anti-bcl-2, and anti-PARP (Cell Signaling, Danvers, MA, USA); anti-ZEB2 (6E5; Active Motif, Tokyo, Japan); anti-cyclin A, anti-bcl-2, anti-VEGF, and anti-phospho-c-Jun (Abcam, Cambridge, MA, USA); anti-ZO-3 (Invitrogen); and anti-E-cadherin and anti-phospho-Tie2 (R&D Systems, Minneapolis, MN, USA). Subcellular fractions were prepared using the Compartmental Protein Extraction Kit (Millipore, Billerica, MA, USA) according to the manufacturer’s instructions.