Figure 4

FANCD2 inhibits tankyrase-1 PARsylation. (A) TNKS1 (3 ug, lanes 1-9) was mixed with increasing amounts of myelin (lanes 2,3), FANCD2 (5 ug, lanes 5-7, 10-12), heat-inactivated FANCD2 (5 ug, lanes 8,9,13) in the presence of [³²P]NAD⁺ (lanes 1-13) and PARP inhibitor 3AB (1 mM, lane 4). After incubation in a PARP buffer, all the reactions were stopped and resulting samples were subjected to SDS-PAGE followed either by autoradiography of the membranes or detected with corresponding antibodies. as described in Experimental section; (B) FANCD2 inhibits tankyrase-1-dependent TRF1 PARsylation. TRF1 protein (3 ug) was added to the reaction mixture as in A. All the reaction products were resolved into SDS PAGE and subjected transfer into nitrocellulose membrane in the buffer containing sodium vanadate. Corresponding membranes were either autoradiographed or probed with anti-FANCD2, anti-TNKS1 or anti-TRF1 antibodies.