Fig. 3: C-terminal region of TWIST1 interacts with BTG2 box B domain. | Cell Death & Disease

Fig. 3: C-terminal region of TWIST1 interacts with BTG2 box B domain.

From: Translational downregulation of Twist1 expression by antiproliferative gene, B-cell translocation gene 2, in the triple negative breast cancer cells

Fig. 3

a In vitro immunoprecipitation (IP) analysis was performed in 293TN cells with anti-HA antibody. Pulldown of BTG2-HA using 1.0 µg of anti-HA antibody showed its interaction with v5-Twist1, depending on its concentration. Normal IgG was employed as a negative control of IP assay. Immunoblotting is the representative of three independent experiments. b Fractionation of the cytoplasm and nucleus of 293TN cells transfected with v5-Twist1 and BTG2-HA was performed, and the interaction of Twist1 and BTG2 was further examined by IP with 1.0 µg of anti-HA antibody. The data suggest that interaction was mainly observed in the nuclear fraction than in the cytoplasm. Star indicates nonspecific band. c In vitro GST-pulldown assay. GST and GST-BTG2 beads were incubated with 293TN cell lysates transfected with v5-Twist1 at 4 °C overnight. The beads were pulled down and subjected to immunoblot analysis with anti-v5 antibody. Note the direct interaction of BTG2 with v5-Twist1. d IP analysis was performed in 293TN cells after transfection with Flag-Twist1 (1.0 μg) and v5-BTG2 (1.0 μg) genes. In vitro interaction of Flag-Twist1 and v5-BTG2 was able to be shown by IP with 1.0 µg of anti-Flag antibody. Immunoblotting is the representative of three independent experiments. e For mapping BTG2 domain bound to Twist1, 293TN cells were transfected with either BTG2-HA wild type or deletion mutants (ΔBox-A, ΔBox B) along with v5-Twist1 gene. IP analysis performed with anti-HA antibody found that overexpression of ΔBox-B mutant significantly reduced BTG2-Twist1 interaction, indicating that box B in BTG2-HA interacted with v5-Twist1 protein. *The reduced interaction between BTG2-HA and v5-Twist1. BTG2 with ΔBox-B also abrogated interaction with endogenous cNOT7. f IP analysis with anti-v5 antibody. Procedure was the same as described in the e. Note the star revealing significant loss of the interaction. g To investigate the domain in Twist1 interacting with BTG2-HA, 293TN cells were transfected with either Twist1 wild type (1–202) and its deletion constructs (1–121, 1–161) along with BTG2-HA. IP performed with anti-HA antibody showed that the wild-type Twist1, but not the deletion mutants, could interact with BTG2-HA. h In vitro interaction analysis by GST-pulldown assay. GST and GST-BTG2 beads were incubated with 293TN cell lysates transfected with either wild type or the deletion constructs of Twist1 gene at 4 °C overnight. When the beads were subjected to immunoblot analysis with anti-Flag antibody, the interaction of BTG2 with wild-type Twist1, but not deletion mutants, was observed. All data clearly indicate the in vitro interactions of C-terminal region of Twist1 with BTG2 box B domain. Blots (b, c, and e–g) are representative of two independent experiments

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