Figure 7
From: A prelude to the proximity interaction mapping of CXXC5
Identification of a sub-region of MeCP2 critical for interacting with CXXC5. (a) Schematics of the wild-type MeCP2 and the carboxyl-terminally truncated MeCP21-400 bearing the 3xFlag (3F) or the HA (HA) tag at the amino-terminus. The N-terminal domain (NTD), methyl CpG binding domain (MBD), inter-domain (ID), transcription repression domain (TRD), and C-terminal domain (CTD) containing a WW protein interaction domain are indicated. (b) The synthesis of MeCP2 (FL) or HA-MeCP21-400 (Δ) in transiently transfected cells was assessed with WB using the HA antibody. EV indicates empty vector as control and MM denotes molecular masses in kDa. (c,d) HEK293 cells were transiently co-transfected with the expression vector bearing cDNA for 3F-CXXC5 and HA-MeCP2 or HA-MeCP21-400. Nuclear extracts were subjected to Co-IP with the HA or the isotype-matched IgG. The precipitates were subjected to WB using the Flag antibody. The membrane was re-probed with the HA antibody. 10% of nuclear extracts was used as input control. Molecular masses (MM) in kDa are indicated. (e) Nuclear extracts of HEK293 cells transiently co-transfected with an expression vector bearing cDNA for the 3F-CXXC domain (3F-CXXC), HA-MeCP2, or HA-MeCP21-400, were subjected to WB using the Flag, HA or HDAC1 antibody. Molecular masses (MM) in kDa are indicated. (f) Nuclear extracts, 500 µg, co-synthesizing CXXC, and HA-MeCP2, or HA-MeCP21-400, were subjected to Co-IP with the HA antibody or the isotype-matched IgG. The precipitates were subjected to WB using the Flag antibody. The membrane was also re-probed with the HA antibody. 10% of nuclear extracts was used as input control. Molecular masses (MM) in kDa are indicated.
