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. 2018 Sep 28;37(24):e99559. doi: 10.15252/embj.201899559

Figure EV2. CDKLs 1–4 cannot phosphorylate MAP1S or CEP131 in cells.

Figure EV2

  1. Schematic representation of CDKLs 1–5. The kinase catalytic domain is highlighted in dark blue. Amino acid numbers at the N‐ and C‐termini are indicated; the positions of conserved residues in the ATP binding sites that were mutated to render these protein “kinase‐dead” are also indicated.
  2. HEK293 cells were co‐transfected with C‐terminally tagged FLAG‐tagged CDKLs 1,2,3,4 or 5 (wild type “WT” or the relevant kinase‐dead mutant) and HA‐MAP1S. Anti‐HA precipitates were subjected to Western blotting with the antibodies indicated. “Hi” higher exposure; “lo” lower exposure. The input extracts were also subjected to immunoblotting (lower panels). Three independent experiments were done, and one representative experiment is shown.
  3. Same as (B) except that HEK293 cells were co‐transfected with C‐terminally tagged FLAG‐tagged CDKLs 1,2,3,4 or 5 (wild type “WT” or the relevant kinase‐dead mutant) and HA‐CEP131. Three independent experiments were done, and one representative experiment is shown.
  4. HEK293 cells were transfected with C‐terminally tagged FLAG‐tagged CDKLs 1,2,3,4 or 5 (wild type “WT” or the relevant kinase‐dead mutant). Anti‐FLAG precipitates were incubated with the MAP1S S900 synthetic peptide in the presence of [γ‐32P]‐labelled ATP‐Mg2+, and peptide phosphorylation was measured by Cerenkov counting. Data are represented as mean ± SEM from three independent experiments.

Source data are available online for this figure.