Gtx104616

Total cell extracts (20 μg of protein) were fractionated by 10–12% SDS–PAGE, transferred to an immobilon polyvinylidene difluoride membrane (Amersham Biosciences, Corston, UK), and probed with one of the specific primary antibodies against PTP4A3 (1:1000, GTX100600, GeneTex, Hsinchu, Taiwan), p-STAT3 (1:1000, GTX104616, GeneTex, Hsinchu, Taiwan), STAT3 (1:1000, GTX11800, GeneTex, Hsinchu, Taiwan), STAT5A (1:1000, GTX113351, GeneTex, Hsinchu, Taiwan), FOXO3A (1:1000, GTX100277, GeneTex, Hsinchu, Taiwan), TERT (1:1500, GTX124242, GeneTex, Hsinchu, Taiwan), TOP2A (1:1500, GTX100689, GeneTex, Hsinchu, Taiwan) p-JAK2 (1:1000, GTX132784, GeneTex, Hsinchu, Taiwan), JAK2 (1:1000, GTX01195, GeneTex, Hsinchu, Taiwan), p-P65 (1:1000, GTX133899, GeneTex, Hsinchu, Taiwan), P65 (1:1000, GTX102090, GeneTex, Hsinchu, Taiwan) and β-actin (1:10,000, #4970, Cell Signaling Technology, Danvers, MA, USA) overnight at 4°C. Subsequently, the membrane was hybridized with appropriate HRP–conjugated secondary antibodies for 1 h at room temperature. Finally, immune complexes were visualized using the chemiluminescence method with an ECL detection kit (Merck Millipore, Temecula, CA, USA) on Fujifilm LAS-3000 (Fujifilm, Tokyo, Japan).