Anti e2f8

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).

A radioimmunoprecipitation assay (RIPA) buffer (Thermo Fisher Scientific Inc., Waltham, MA, USA) was used to extract proteins. We measured the protein concentrations using the Pierce BCA Protein assay kit (Thermo Fisher Scientific). The proteins were boiled with 2× sample buffer and were subsequently resolved on 10% sodium dodecyl sulfate-polyacrylamide gels before being electrophoretically transferred to polyvinylidene difluoride membranes (Millipore, Billerica, MA, USA). After blocking with 5% nonfat dried milk in 1× Tris-buffered saline containing 0.1% Tween 20 (pH 7.6) (TBST) at room temperature for 1 h, the membranes were incubated with anti-β-catenin, anti-Notch1, anti-HES1, anti-P300, anti-E-cadherin, anti-N-cadherin, anti-Vimentin, anti-Snail, (all from Cell Signalling Technologies, Danvers, MA, USA), anti-E2F8, anti-Wnt5β, and anti-Twist (all from Abcam, Cambridge, MA) antibodies overnight at 4 ℃. Anti-β-actin antibody (Sigma-Aldrich) was used as an internal control. Membranes were washed with TBST and incubated with horseradish peroxidase-conjugated secondary antibodies (Jackson Immunoresearch, West Grove, PA, USA) for 1 h at room temperature. After washing again with TBST, signal was detected using an enhanced chemiluminescence kit (Thermo Scientific, Rockford, IL, USA), and intensity was quantified using ImageJ software.