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. 2023 Jan 19;24(3):2033. doi: 10.3390/ijms24032033

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© 2023 by the authors.

Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/).

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PP5 dephosphorylates Plk4, in vitro and in vivo. (a) In vitro dephosphorylation of p-MBP-Plk4-TE (“p-” refers to the self-phosphorylated, while “TE” refers to the T172E activating mutation in Plk4, respectively) was tested by gel-shift assay. The electrophoretic mobility of the phosphorylated forms is decreased, therefore they run higher, while the dephosphorylated species run lower in a long preparative SDS-PAGE. Purified recombinant p-MBP-Plk4-TE is efficiently dephosphorylated by the λ-phosphatase (λPPase) and GST-PP5, in vitro. GST-PP5^H326N serves as the negative control; “ctrl” refers to non-treated purified kinase. (b) The GST-PP5-mediated dephosphorylation kinetics of p-MBP-Plk4-TE is presented by Coomassie brilliant blue-stained preparative SDS-PAGE. Incubation times (minutes) are indicated. λPPase serves as the positive control; “ctrl” refers to non-treated sample. (c) In vivo dephosphorylation of Plk4 in bacteria was tested by gel-shift assay. MBP-Plk4-TE or its kinase dead (TEKM) version were co-expressed with His₆-PP5 or its inactive form, His₆-PP5^H326N, respectively, in bacteria. Crude cell lysates were analyzed by SDS-PAGE, which shows that MBP-Plk4-TE dephosphorylation requires the active His₆-PP5, in vivo. MBP-Plk4-TEKM is not capable of self-phosphorylation. His₆-PP5^H326N is unable to dephosphorylate Plk4.

PP5 dephosphorylates Plk4, in vitro and in vivo. (a) In vitro dephosphorylation of p-MBP-Plk4-TE (“p-” refers to the self-phosphorylated, while “TE” refers to the T172E activating mutation in Plk4, respectively) was tested by gel-shift assay. The electrophoretic mobility of the phosphorylated forms is decreased, therefore they run higher, while the dephosphorylated species run lower in a long preparative SDS-PAGE. Purified recombinant p-MBP-Plk4-TE is efficiently dephosphorylated by the λ-phosphatase (λPPase) and GST-PP5, in vitro. GST-PP5^H326N serves as the negative control; “ctrl” refers to non-treated purified kinase. (b) The GST-PP5-mediated dephosphorylation kinetics of p-MBP-Plk4-TE is presented by Coomassie brilliant blue-stained preparative SDS-PAGE. Incubation times (minutes) are indicated. λPPase serves as the positive control; “ctrl” refers to non-treated sample. (c) In vivo dephosphorylation of Plk4 in bacteria was tested by gel-shift assay. MBP-Plk4-TE or its kinase dead (TEKM) version were co-expressed with His₆-PP5 or its inactive form, His₆-PP5^H326N, respectively, in bacteria. Crude cell lysates were analyzed by SDS-PAGE, which shows that MBP-Plk4-TE dephosphorylation requires the active His₆-PP5, in vivo. MBP-Plk4-TEKM is not capable of self-phosphorylation. His₆-PP5^H326N is unable to dephosphorylate Plk4.