Figure 2. Identification of the PARP1 Binding Site in TIMELESS.
(A) HEK293T cells were transfected with either an empty vector (EV), FLAG-tagged TIMELESS, or FLAG-tagged TIMELESS mutants as indicated. Asterisks indicate the insertion of a STOP codon after the numerically specified codon. 24 hr post-transfection, WCEs were subjected to IP with α-FLAG resin and immunoblotted as indicated.
(B) Schematic representation of TIMELESS mutants tested for binding to PARP1. TIMELESS mutants that interacted with endogenous PARP1 are designated with the symbol (+); those unable to co-precipitate PARP1 are designated with the symbol (−).
(C) The experiment was performed as in (A), except that different TIMELESS mutants were used.