Anti ha clone 3f10

HEK 293 cells were seeded into 10-cm dishes and transfected with CS2 + or TAF1 expression constructs and incubated for 24–36 h at 37 °C. Before harvesting, cells were cross-linked with 400 μl of 37% formaldehyde and analyzed as previously described^{57 (link)}. In brief, cells were lysed in 0.5 mL immunoprecipitation (IP) buffer (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 5 mM EDTA, 1% Triton X-100, and 0.5% Nonidet P-40) containing protease inhibitors and sonicated 8 times for 20 s at 40% amplitude (Branson Digital Sonifier). Nuclear extract was incubated overnight at 4 °C with either anti-HA (clone 3F10, Sigma Aldrich) or mouse IgG (Abcam). Immunoprecipitated complexes were washed and eluted. Cross-links were reversed and DNA was isolated. Input DNA was purified from 100 μl extract. Fifty nanograms of input DNA and 2 μL of purified TAF1 bound DNA was amplified with SsoFast EvaGreen Supermix (Bio-Rad) using primers spanning the promoters of cyclin D1 and cyclin A2 (Supplemental Table S1). Quantitative PCR was performed on the Applied Biosystems 7500Fast Real-Time PCR system and the data expressed as percent input.

Anti-HA (clone 3F10, Sigma-Aldrich), anti-α-Tubulin (clone DM1A, Sigma-Aldrich), anti-Cdk1 (ab18, Abcam, Cambridge, UK), anti-ERK1/ERK2 (9102, Cell Signaling, Danvers, MA), anti-xGLD2 (rabbit polyclonal antibody raised against KRRSDEGNSPYDVKC peptide, X. laevis), anti-4E-T (2297, Cell Signaling), anti-DDX6 (PD009, MBL, Nagoya, Japan), anti-hCPEB4 for WB (rabbit polyclonal antibody raised against amino acids 1–302), anti-hCPEB4 for IP (ab83009, Abcam), anti-phospho Histone H3 (Ser 10) (06–570, Merck Millipore, Billerica, MA), anti-Symplekin (610644, BD Transduction Laboratories, Franklin Lakes, NJ), anti-CPSF2 (ab126760, Abcam), anti-PARN (ab89831, Abcam), anti-hGLD2 (AP5092, Abgent, San Diego, CA), anti-S6 (2317, Cell Signaling), anti-Vinculin (ab18058, Abcam).