Figure EV1. Mutations in the IN1 bNLS linker sequence abolish the interaction with AC40.

1. Validation of the expression of GAD‐IN1 fusion proteins for the two‐hybrid assay shown in Fig 1B. Whole‐cell extract samples were prepared for immunoblotting from 1 DO₆₀₀ of the cell culture by TCA precipitation. GAD‐IN1 fusion proteins were detected by Western blot with anti‐HA antibody (12CA5, Roche). All constructs harbor an HA‐tag that is present between the GAD and IN1 sequences in the original pACTII vector. Expected sizes of the proteins are 26 kDa for GAD‐IN1_578–635 and GAD‐IN1_578–635 mutants and 82 kDa for GAD‐IN1_1–578. “x”, non‐relevant sample.
2. Growth control of cell cultures corresponding to the two‐hybrid assay shown in Fig 1B. Twofold serial dilutions of cell cultures, starting from 10⁻¹, were plated on DO‐Leu‐Trp plates to check for growth.
3. Growth control of cell cultures corresponding to the two‐hybrid assay shown in Fig 1C. Twofold serial dilutions of cell cultures, starting from 10⁻¹, were plated on DO‐Leu‐Trp plates to check for growth.
4. Validation of the expression of GAD‐IN1 fusion proteins for the two‐hybrid assay shown in Fig 1C. Whole‐cell extract samples were prepared for immunoblotting from 1 DO₆₀₀ of the cell culture by TCA precipitation. GAD‐IN1 proteins were detected by Western blot with anti‐GAD antibody (Santa Cruz Biotechnology). Expected sizes of the proteins are 82 kDa for GAD‐IN1_1–578, 26 kDa for GAD‐IN1_578–635, and 24 kDa for GAD‐IN1_596–630. “x”, non‐relevant sample.

Source data are available online for this figure.