Sc 293059

Cells were harvested at the indicated times and rinsed twice with PBS. Cell extracts were prepared with lysis buffer and centrifuged at 13,000 g for 10 min at 4 °C. Protein samples (100 μg) were electrophoresed using 10 % polyacrylamide gels and transferred to PVDF membranes. After the membranes were blocked with 5 % BSA for 1 h at room temperature, they were incubated with FOXD3 antibody (ab67758, 1:1,000, Abcam, Cambridge, MA, USA), STAT3 antibody (#4904, 1:2,000, Cell Signaling, Danvers, MA, USA), p-STAT3(Tyr705) antibody (sc-8059, 1:1,000, Santa Cruz, Dallas, TX, USA), p-STAT3(Ser727) antibody (sc-293059, 1:1,000, Santa Cruz, Dallas, TX, USA), Flag antibody (ab49969, 1:3,000, Abcam, Cambridge, MA, USA) or HA antibody (ab18230, 1:3,000, Abcam, Cambridge, MA, USA) in 5 % BSA overnight at 4 °C. Secondary antibodies (1:3,000) were labeled with horseradish peroxidase (HRP). The signals were checked by autoradiography film when HRP substrate was added to the membranes.