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. Author manuscript; available in PMC: 2021 Aug 25.
Published in final edited form as: Cell Rep. 2021 Aug 10;36(6):109514. doi: 10.1016/j.celrep.2021.109514

Figure 5. CycK/CDK13 phosphorylates SERINC5 at S360.

Figure 5.

(A) SERINC5 was expressed with the indicated CDK13 or CDK12 proteins in the presence of WT or ΔNef HIV-1 in HEK293T cells. SERINC5, CDK13, and CDK12 proteins were detected by WB using an anti-FLAG antibody.

(B) SERINC5 was expressed with WT or ΔNef HIV-1 in HEK293T cells and treated with THZ531 at 100 μM. SERINC5 expression was analyzed by WB using an anti-FLAG antibody.

(C) SERINC5 and SERINC5S360A were expressed with WT or ΔNef HIV-1 in HEK293T cells and purified similar to as previously. After SDS-PAGE, proteins were analyzed after being stained with Coomassie brilliant blue or Phos-tag.

(D) GST-ICL4 and GST-ICL4S360A recombinant proteins were expressed in E. coli and purified by glutathione resin. An in vitro kinase assay was conducted by incubating these proteins with affinity-purified CycK/CDK13. After SDS-PAGE, proteins were analyzed after being stained with Coomassie brilliant blue or Phos-tag.

(E) Peptide 357FCFSPGGEDTEEQQPGK373, its S360-phosphorylated version, and its T366K-substituted version were synthesized. They were analyzed by high-resolution MS via direct infusion or analyzed by CZE-MS after treatment with affinity-purified CycK/CDK13.