Western Blot Analysis of IGF-2 and Phospho-Akt

Cultured cells and tumor samples were processed for western blot as previously described [10 (link)], except the membrane was incubated in 1 M DTT solution at room temperature for 5 min then washed with TBS-T three times before adding IGF-2 antibody. Phosphatase inhibitors (P2850, P5726, Millipore-Sigma, Burlington, MA, USA) were added to the lysis buffer for P-Akt. The antibodies used were IGF-2, 8H1, MA5-17096, 1:1000, ThermoFisher Scientific, Waltham, MA, USA, α-tubulin, Ab7291, 1:10,000, Abcam, Cambridge, UK, GAPDH, sc-47,724, 1:1000, Santa Cruz Biotechnology, Dallas, Texas, USA, Akt, 2938, P-Akt, 4058, 1:1000, secondary antibodies 7074 and 7076 S, 1:10,000, Cell Signaling Technology, Danvers, MA.