P igf1r

After the treatment cells were re-suspended in lysis buffer (50 mM Tris-base [pH 7.5], 0.5M NaCl, 1 mM b-mercaptoethanol, 1% NP-40, 1% glycerol and protease inhibitor tablets). Samples containing equal amounts of total protein (40 μg) were separated by 10% SDS-PAGE and then transferred onto polyvinylidene difluoride membranes (Millipore Corp., Billerica, MA, USA). Residual protein sites were blocked in 1 × Tween-20/Tris-buffered saline (1 × TBS) containing 5% skim milk. Then, the membranes were incubated with 1:1000 diluted primary antibody solutions in 1 × TBS buffer at 4°C for at least 1 overnight, depending affinity of each antibody. After the primary antibody solutions were recycled, blots were incubated with secondary antibody solutions 1 × TBS for 1 h.
Primary antibodies against b-actin, p-Akt, Bax, b-catenin, caspase-3, caspase-9, COL2A1, CTGF, cytochrome C, p-IGF1R, MMP2, MMP9, p-PI3K, p-Smad2/3, SP1, TIMP-1, TIMP-2, tPA and uPA were purchased from Santa Cruz Biotechnology (Dallas, TX, USA). Primary antibody against insulin-like growth factor-1 (IGF-1) was purchased from Abcam Plc. (Cambridge, United Kingdom). Secondary antibodies used in this study (anti-goat-HRP, anti-mouse-HRP and anti-rabbit-HRP) were purchased from Santa Cruz Biotechnology (Dallas, TX, USA).