Detect ARTD10-Mediated PKCδ MARylation

HEK293 cells co-expressing EGFP-PKCδ and HA-ARTD10-WT were treated with 3 μM OUL35 or the appropriate volume of DMSO as vehicle control for 16 h. Forty-eight hours post transfection, cells were lysed in RIPA buffer (10 mM Tris, pH 7.4; 150 mM NaCl; 1% NP-40; 1% DOC; 0.1% SDS; protease inhibitor cocktail, Sigma) including 10 μM Olaparib (Selleckchem) and the lysates were cleared at 4 °C for 30 min. EGFP-PKCδ was immunoprecipitated with 5 μl of GFP-TRAP magnetic agarose beads (Chromotek) at 4 °C for 1 h. Afterwards, the beads were washed three times in RIPA buffer. Samples were fractionated by SDS-PAGE and transferred to nitrocellulose membranes to visualize MARylation using an anti-PAR/MAR antibody (Cell Signaling Technology, #83732). The individual proteins were analyzed using anti-GFP antibodies (Rockland, mouse monoclonal 600-301-215 M) to visualize EGFP-PKCδ, anti-α-Tubulin (Sigma, #T5168), anti-ARTD10 [6 (link)], goat-anti-rabbit-HRP (Jackson Immunoresearch, #111-035-144), and goat-anti-mouse-HRP (Jackson Immunoresearch, #115-036-068).