Co-immunoprecipitation and western blot

For MCF-7 cells expressing YFP and NLS-YFP-Pfn1(WT and S137D), nuclear extracts were prepared using a modified Stillman protocol (Méndez and Stillman, 2000). Briefly, approximately 5 × 10⁶ cells were lysed with 200 μl hypotonic buffer A (10 mM HEPES, pH 7.9,10 mM KCl, 1.5 mM MgCl2, 0.34 M Sucrose, 10% Glycerol, 1 mM DTT, 0.1% Triton X-100, Protease and phosphatase inhibitor cocktails) for 15min on ice, and centrifuged at 1,300 g for 5 min. After removing cytosol, nuclei were washed once with buffer A without Triton X-100, and lysed with 200 μl nuclear buffer (10mM HEPES, pH 7.0, 200mM NaCl, 1mM EDTA, 0.5% NP-40, and protease-inhibitor cocktail) for 10 min on ice followed by centrifugation at 13,000 g for 10 min. Clarified nuclear lysates were incubated with 2 μg of the GFP antibody (DSHB, #12E6) overnight at 4°C with rotation and mixed with 30 μL of the protein G beads (CST, #9007S) at 4°C for 1-2 hr. The beads were then washed 3-5 times with IP buffer (40 mM Tris-Cl, pH 7.5, 150 mM NaCl, 2mM EDTA, 0.5% Triton X-100,10% glycerol, and 1 mM DTT), and heated at 95°C in SDS sample buffer for 5 min. For parental and shLUC or shENL-infected MCF-7 cells, approximately 5 × 10⁶ cells were lysed with 800μl RIPA buffer (EMD Millpore, 20-188) and centrifuged at > 15,000 g for 10 min. 350 μl of clarified lysates were incubated for 3 hr at 4°C with 20 μL of protein G magnetic Dynabeads (Thermo Fisher, 10004D), and 3 μg of normal IgG, ENL, or Cyclin T1 antibodies. Beads were washed 6 times with cold PBS + 0.02% Tween-20, followed by heating in SDS sample buffer. All samples were subsequently analyzed by SDS-PAGE and western blot analysis.

For analyzing proteins in cell lysates by western blot, lysates were prepared using RIPA buffer (CST, #9806) supplemented with protease and phosphatase inhibitors and clarified by centrifugation at 15,000 g for 10min. After normalization using the Quick Start Bradford 1x Dye Reagent (Bio-Rad, #5000205), 1-10 μg denatured proteins were analyzed by SDS-PAGE and transferred to nitrocellulose membrane (Santa Cruz, sc-3718). Primary antibodies were incubated overnight at 4°C in TBS/0.1% Tween-20 containing 5% bovine serum albumin. Secondary antibodies were incubated at room temperature for 1-2 hr. Proteins were developed using the SuperSignal West Dura Extended Duration Substrate (34075, ThermoFisher) or West Femto Maximum Sensitivity Substrate (34096, ThermoFisher), and imaged on a Gel Doc XR imaging system (Bio-Rad). Band intensities were quantified by ImageLab.