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. 2021 Sep 2;11:17587. doi: 10.1038/s41598-021-97060-6

Figure 3.

Figure 3

Interaction of MAZ and CXXC5. (a) Schematics of the full length (MAZ) or the amino-terminally truncated MAZ (MAZΔN) variant bearing a Flag or HA tag at the amino-terminus. Ovals in orange color indicate the C2H2-type zinc finger domains. (b) To evaluate the protein synthesis, HEK293 cells were transfected with the expression vector bearing none (EV), the HA-MAZ, HA-MAZΔN, or 3F-CXXC5 cDNA. Nuclear extracts were subjected to SDS-10%PAGE followed by WB using the HA or the Flag antibody. (c) To assess the intracellular localization of the proteins, HEK293 cells were transiently transfected for 48 h with the expression vector bearing the 3F-CXXC5 and the HA-MAZ or HA-MAZΔN cDNA. Cells were then subjected to ICC using the Flag (green channel) or the HA (red channel) antibody. DAPI staining indicates the nucleus. The scale bar is 10 μm. (d,e) To assess the co-synthesis of proteins, the nuclear extracts of HEK293 cells, transiently transfected with the expression vector bearing 3F-CXXC5 and/or (d) the HA-MAZ or (e) the HA-MAZΔN cDNA, were subjected to WB analyses. Proteins were immunoblotted (IB) with the Flag or HA-antibody. HDAC1 used as a loading control was probed with the HDAC1 antibody. (f,g) The nuclear extracts, 500 µg, of transiently co-transfected HEK293 cells were subjected to Co-IP with the HA or the isotype-matched IgG. 10% of nuclear lysate was used as input control. The precipitates were subjected to SDS-10%PAGE followed with WB using the Flag (f) or the HA (g) antibody. Molecular masses (MM) in kDa are indicated.