1. Validation of novel CARM1 substrates.
Validation of novel CARM1 substrates. (A, E, F) Whole cell lysates from CARM1 WT (+/+) and KO (−/−) MEFs were immunoprecipitated with antibodies against MED12, SRC-1, or SRC-3 proteins, and the eluted samples were subjected to Western blotting with αADMACARM1 and αH3R17me2a antibodies. αMED12, αSRC-1, and αSRC-3 blots (bottom panels in A, E, F) demonstrate equal expression of these proteins in CARM1 WT (+/+) and KO (−/−) MEFs. (B, C) MCF-7-Tet-on-shCARM1 cells were untreated or treated with doxycycline (1 μg/ml) for 8 d and transiently transfected with FLAG-KMT2D and -GPS2, separately. Total cell lysates were immunoprecipitated with αFLAG antibody, and the eluted samples were subjected to Western blotting with αADMACARM1 and αFLAG antibodies. (D) HeLa cells were transiently transfected with FLAG control, FLAG-KMT2Da, or FLAG-KMT2Da-R3727K. Total cell lysates were immunoprecipitated with αFLAG antibody, and the eluted samples were subjected to Western analysis with αADMACARM1 and αFLAG antibodies. αFLAG blot of the input samples shows equal expression of the WT and mutant KMT2Da proteins. KMT2Da represents a fragment (3,619–4,285 aa) of the full-length KMT2D protein (NP_003473).
Donghang Cheng et al. LSA 2018;1:e
© 2018 Cheng et al.