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. 2023 Jan 13;13(1):e1153. doi: 10.1002/ctm2.1153

FIGURE 8.

FIGURE 8

TRIP12 interacts with TAF10 and inhibits MYC activity. (A and B) Flag‐labelled TAF10 and GFP‐labelled TRIP12 were transfected into MCF7 cells. Cells were treated with Z363 (7.5 μg/ml) for 24 h, and the interaction between TAF10 and TRIP12 was detected by Co‐IP. (C) Endogenous TRIP12 binds to TAF10 and vice versa. MCF7 cell lysates were subjected to immunoprecipitation using IgG, anti‐TAF10 or anti‐TRIP12 antibodies and then analysed by Western blotting as indicated. (D) The interaction between TRIP12 and TAF10 was detected by GST pulldown assay in vitro. (E) Interaction between the TAF10 mutants and TRIP12 in MCF7 cells was detected using Co‐IP. (F) Interaction between the TRIP12 mutants and TAF10 in MCF7 cells was detected using Co‐IP. (G) HA‐ub, Flag‐TAF10 and GFP‐TRIP12 plasmids were transfected alone or co‐transfected into MCF7 cells, and the ubiquitination of TAF10 was detected by Co‐IP. (H) Ubiquitination of TAF10 was analysed in WT and siTRIP12 MCF7 cells. (I) Lys‐48‐linked ubiquitination catalysed by the wild‐type TRIP12 (GFP‐TRIP12‐WT) was further confirmed by seven Lys‐only ubiquitin mutants, Lys‐6, ‐11, ‐27, ‐29, ‐33, ‐48 and ‐63. Blots were representative of three independent experiments.