Co-Immunoprecipitation of HEK293T Cell Proteins

HEK 293T whole cell extracts for co-IP were prepared using lysis buffer containing 1%TX-100, 50 mM HEPES (pH 7.4), 138 mM KCl, 4 mM NaCl, 50 mM sodium pyrophosphate, 100 mM sodium fluoride, 10 mM EDTA, 1 mM EGTA pH 8, 50 μM PMSF, 1 mM β-Glycerophosphate, 1 mM sodium orthovanadate, and protease inhibitors (Thermo Scientific cat # 88265) as previously described (Lamia et al., 2004). Immunoprecipitation was performed using anti-Flag M2 agarose beads (Sigma cat #A2220). Antibodies for Western Blots were anti-FLAG polyclonal (Sigma cat #F7425), anti-V5 polyclonal (Bethyl Labs cat# A190–120A), anti-HA polyclonal (Sigma cat # H6908), anti-α-TUBULIN (Sigma cat # T5168), anti-E2F1 (Santa Cruz KH95), anti-E2F8 (Abcam ab109596), CRY1-CT and CRY2-CT^{11 (link)}. For CHX assay, HEK293T cells were lysed with a RIPA Buffer containing 1% TX-100, 147 mM NaCl, 12 mM Sodium deoxycholate, 0.1% SDS, 50 mM Tris pH 8.0, 10 mM EDTA, 50 μM PMSF, 1 mM β-Glycerophosphate, 1 mM Sodium Orthovanadate and protease inhibitors (Thermo Scientific cat # 88265).