Cwc15, Syf2, and Snt309 are needed for the interaction between TREX and Prp19C. (A) Coomassie gel of EGTA eluates from cells expressing Hpr1-TAP and HA-tagged Syf1 in a WT, Δcwc15, Δisy1, Δntc20, Δsyf2, and Δsnt309 background (upper panel). CBP, Calmodulin-binding protein; the faster migrating band of Hpr1-CBP in Δcwc15 and Δsyf2 cells was verified by mass spectrometry and is indicated by a star. Syf1-HA levels of extracts (Input) and eluates (IP) were assessed by western blotting using antibody against the HA-tag (lower panels). A strain expressing Syf1-HA only (i.e., lacking the TAP tag on Hpr1) served as negative control. (B) Quantification of western blots of three independent copurifications as shown in A. The intensity of the western band of copurified Syf1 was normalized to the amount of purified Tho2. (C) Coomassie gel of EGTA eluates of purified, N-terminally TAP-tagged Hpr1 from WT, Δcwc15, Δsyf2, and Δcwc15Δsyf2 cells (upper panel) and western blots assessing the corresponding Syf1-HA levels of extracts (Input) and eluates (IP) (lower panels). (D) Quantification of Syf1 copurified with TREX and normalized to Tho2 of three independent experiments as shown in C.
