Anti tcea1

RNA-ChIP was performed using the EZ-Magna RIP kit (Millipore) per the manufacturer's instructions as previously described. In brief, 10⁷ cells were lysed with RIP lysis buffer with one freeze-thaw cycle. Cell extracts were coimmunoprecipitated with anti-TCEA1, anti-RBMX, anti-MCM2 or anti-CBX3 (Abcam), and the retrieved RNA was subjected to real-time qPCR analysis. Normal rabbit IgG was used as a negative control.

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 30 min at 4°C. Protein samples were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) in 7.5% (w/v) polyacrylamide gels and transferred to polyvinylidene fluoride membranes. After blocking with 5% BSA for 1 h at room temperature, the membrane was incubated with 2.5 μg/ml antibody in 5% BSA overnight at 4°C. The membrane was then incubated with a secondary antibody conjugated to a fluorescent tag (Invitrogen). The band signals were visualized and quantified using the Odyssey Infrared Imagining System (LI-COR, USA). The following antibodies were used in this study: Anti-GALNT8 (Abcam, USA), Anti-TCEA1(Abcam, USA) and GAPDH (Sigma-Aldrich).