Gfp trap dynabeads

LNCap cells (ATCC, CRL‐1740) were cultured at 37°C with 5% CO₂ air in Roswell Park Memorial Institute (RPMI) 1640 medium supplemented with 10% fetal bovine serum (FBS). Authenticity was confirmed by STR profiling. Cells were tested and confirmed negative for mycoplasma contamination. Cells were transfected with ELAC2 wild‐type or ELAC2 A541T with EGFP fused to the C‐termini using FuGene (Promega) and incubated for 72 h prior to lysis in 260 mM sucrose, 100 mM KCl, 20 mM MgCl₂, 10 mM Tris–HCl (pH 7.5), 1% digitonin and complete EDTA‐free protease inhibitor cocktail for 30 min at 4°C. Lysates were incubated with GFP‐Trap Dynabeads (Chromotek) for 2 h at 4°C. Beads were washed in digitonin‐wash buffer (same components as lysis buffer except with 0.1% digitonin), washed again in wash buffer without digitonin and then partially digested in 50 mM Tris–HCl pH 7.5, 1 mM TCEP and 5 mM CAA supplemented with 75 ng Sequencing Grade Modified Trypsin (Promega) for 30 min at room temperature. The beads were removed using DynaMag‐2 magnet (Invitrogen), and eluates were incubated at 37°C overnight for complete digestion followed by preparation for label‐free mass spectrometry.

The MARylation activity assay was performed as described in [7 (link)]. Briefly, clarified lysates of HEK-293 transfected with the different Parp6 expression constructs were immunoprecipitated with GFP-trap dynabeads (Chromotek, Planegg-Martinsried, Germany) for 1 h at 4 °C with rotation. Beads were washed three times with lysis buffer (50 mM HEPES pH 7.4 + 150 mM NaCl + 1 mM MgCl₂ + 1% triton x-100) and incubated with alkyne NAD⁺ in the presence of 3 μM veliparib (Selleckchem, Pittsburgh, PA, USA) to inhibit PARylation by PARP1/2 for 2 h at RT, with shaking. Beads were washed two times with PARP reaction buffer (PRB buffer) and once with PBS. Click reaction was performed for 1 h at RT in PBS containing 1 mM CuSO₄, 100 µM TBTA, 100 µM biotin, and 1 mM TCEP. Proteins were then eluted by boiling the beads for 5 min in 1.5x Laemmli buffer, separated by 10% SDS-PAGE, and transferred to nitrocellulose membranes (Amersham Protran 0.45 NC, Millipore Sigma, St. Louis, MO, USA). Blots were probed with HRP-strepavidin (MARylation) and anti-GFP antibody (normalization), and developed using West Pico or West Femto substrates (Pierce, Thermo Fisher Scientific, Waltham, MA, USA). Blots were then imaged and analyzed (densitometry) using a ChemiDoc system (BioRad, Hercules, CA, USA). List of antibodies used can be found in Table S2.

To determine the interaction between FOXD3 and SUV39H1/H2 and SETDB1, GFP‐SUV39H1 and GFP‐SUV39H2 expressing Suv39h dn ES cells and Foxd3 cKO cells expressing GFP‐FOXD3 expressing Foxd3 cKO cells were used. Cells were subjected to immunoprecipitation with GFP‐trap Dynabeads (Chromotek gtdk‐20) following the manufacturers' protocol. The eluted IP samples were immunoblotted with specific antibodies using established protocols (Bulut‐Karslioglu et al, 2012 (link)). The antibodies used for immunoblot are as follows: Foxd3: Merck Millipore, Catalog number AB5687, 1:500. GFP: Invitrogen, Catalog number A11122, 1:500. Setdb1: Thermo Fisher, Catalog number MA5‐15722, 1:500. Suv39h1: Sigma‐Aldrich (Merck), Catalog number 05‐615, 1:250.