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. 2021 Jun 25;297(2):100921. doi: 10.1016/j.jbc.2021.100921

Figure 2.

Figure 2

Phosphorylation status of TDP1 modulates its interaction with LigIII.A, Upper panel, input (10%) of Flag-TDP1, Flag-TDP1 incubated with λ phosphatase, Flag-TDP1 incubated with phosphatase buffer, GST-TDP1 (Lanes 1–4) and LigIII-DBD and LigIIIβ (lanes 5 and 6). Pull-down assay with Ni-NTA liganded by LigIIIβ (10 pmol, middle panel) and LigIII-DBD (10 pmol, lower panel). Lanes 1 to 4, immunoblotting with rabbit anti-TDP1-C-term (Abcam) and lanes 5 and 6, with mouse anti-6His mAb (Abcam). B, identification of phosphorylation sites in insect-cell-expressed TDP1 by mass spectrometry. In the bar diagram, peptide spectrum matches (PSM) represents the total number of times a peptide containing the indicated phosphorylated amino acid was identified. Sites with PSM values greater than 3 are shown. Amino acid residues indicated in red were substituted in subsequent studies. C, Upper panel, input (10%) of the indicated proteins (1 pmol of each). Lower panel, Ni-NTA beads liganded by LigIIIβ (10 pmol) were used to pull down the indicated proteins (10 pmol of each). Immunoblotting with rabbit anti-TDP1-C-term (Abcam).