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. 2023 Aug 2;24(10):e53408. doi: 10.15252/embr.202153408

Figure 2. “Closed” MAD2 interacts with monoamine transporter C‐termini at MAD2 interaction motifs.

Figure 2

  • A
    Glutathione S‐transferase (GST)‐tagged serotonin transporter C‐terminus and the derivative RII‐AAA mutant were purified and used in GST‐protein‐based interaction assays.
  • B–D
    GST pull‐down experiments were conducted in duplicates using the indicated GST fusion proteins. Eluates (30%) were subjected to immunoblotting together with an aliquot (1%) of the input. Blots were stained with Ponceau S, and MAD2 was detected by immunoblotting. Densitometric signals were quantified using ImageJ software. Results were compared using unpaired two‐tailed t‐tests; ****P < 0.0001, ns—not significant; exact P‐values are indicated; error bars represent SD of eight independent biological replicates (n = 8).
  • E
    Total cell lysates (5 μg) of indicated transfections were subjected to immunoblotting, using antibodies against YFP and MAD2.
  • F–H
    YFP‐hMAD2 constructs were generated as outlined under “Materials and Methods.” Whole‐cell lysates of transfected HEK‐293 cells were used for GST pull‐down experiments. One‐way ANOVA with Dunnett's multiple post hoc comparison; ***P < 0.001, ns—not significant; error bars represent SD of three independent biological replicates (n = 3).

Source data are available online for this figure.