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Proteintech human plk3 protein

FIG. 1. VRK1 is phosphorylated in Ser342 by <t>Plk3.</t> (A) Sequence in the C-terminal region of VRK1 containing a conserved sequence that is a potential target for Plks. (B) In vitro kinase assay to determine the phosphorylation order between Plk3 and VRK1 and the residue phosphorylated. Plk3 and human VRK1 were expressed in E. coli and puriﬁed for the in vitro kinase assay. KE, K179E substitution in VRK1 that is kinase dead. The phosphorylated proteins were detected by autoradiography (top), and the proteins present in the assay are shown by Coomassie blue staining (bottom). (C) In vitro kinase assay to demonstrate that VRK1 does not phosphorylate kinase-dead Plk3. pGEX-GST-VRK1, pGEX-GST-p53 (1-82), and pGEX-GST-Plk3K52R were expressed in E. coli. The assay was carried out with a constant amount of GST-VRK1 and increasing amounts (as indicated) of GST-Plk3. GST-p53 was used as a positive control. The phosphorylated proteins were detected by autoradiography (top), and the proteins present in the assay are shown by immunoblotting (middle) or Ponceau staining (bottom). (D) Phos- phorylation of VRK1 by transfected Plk3. HEK293T cells were transfected with pCEFL-GST-Plk3 (5 g), and cell extracts were used for immunoprecipitation with monoclonal anti-Flag antibody (control) or monoclonal anti-GST antibody (Plk3). The immunoprecipitates were used for kinase assays using pGEX-GST-VRK1K179E as a substrate.

Human Plk3 Protein, supplied by Proteintech, used in various techniques. Bioz Stars score: 93/100, based on 9 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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human plk3 protein - by Bioz Stars, 2026-06

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1) Product Images from "Plk3 Interacts with and Specifically Phosphorylates VRK1 in Ser 342 , a Downstream Target in a Pathway That Induces Golgi Fragmentation"

Article Title: Plk3 Interacts with and Specifically Phosphorylates VRK1 in Ser 342 , a Downstream Target in a Pathway That Induces Golgi Fragmentation

Journal: Molecular and Cellular Biology

doi: 10.1128/mcb.01341-08

FIG. 1. VRK1 is phosphorylated in Ser342 by Plk3. (A) Sequence in the C-terminal region of VRK1 containing a conserved sequence that is a potential target for Plks. (B) In vitro kinase assay to determine the phosphorylation order between Plk3 and VRK1 and the residue phosphorylated. Plk3 and human VRK1 were expressed in E. coli and puriﬁed for the in vitro kinase assay. KE, K179E substitution in VRK1 that is kinase dead. The phosphorylated proteins were detected by autoradiography (top), and the proteins present in the assay are shown by Coomassie blue staining (bottom). (C) In vitro kinase assay to demonstrate that VRK1 does not phosphorylate kinase-dead Plk3. pGEX-GST-VRK1, pGEX-GST-p53 (1-82), and pGEX-GST-Plk3K52R were expressed in E. coli. The assay was carried out with a constant amount of GST-VRK1 and increasing amounts (as indicated) of GST-Plk3. GST-p53 was used as a positive control. The phosphorylated proteins were detected by autoradiography (top), and the proteins present in the assay are shown by immunoblotting (middle) or Ponceau staining (bottom). (D) Phos- phorylation of VRK1 by transfected Plk3. HEK293T cells were transfected with pCEFL-GST-Plk3 (5 g), and cell extracts were used for immunoprecipitation with monoclonal anti-Flag antibody (control) or monoclonal anti-GST antibody (Plk3). The immunoprecipitates were used for kinase assays using pGEX-GST-VRK1K179E as a substrate.

Figure Legend Snippet: FIG. 1. VRK1 is phosphorylated in Ser342 by Plk3. (A) Sequence in the C-terminal region of VRK1 containing a conserved sequence that is a potential target for Plks. (B) In vitro kinase assay to determine the phosphorylation order between Plk3 and VRK1 and the residue phosphorylated. Plk3 and human VRK1 were expressed in E. coli and puriﬁed for the in vitro kinase assay. KE, K179E substitution in VRK1 that is kinase dead. The phosphorylated proteins were detected by autoradiography (top), and the proteins present in the assay are shown by Coomassie blue staining (bottom). (C) In vitro kinase assay to demonstrate that VRK1 does not phosphorylate kinase-dead Plk3. pGEX-GST-VRK1, pGEX-GST-p53 (1-82), and pGEX-GST-Plk3K52R were expressed in E. coli. The assay was carried out with a constant amount of GST-VRK1 and increasing amounts (as indicated) of GST-Plk3. GST-p53 was used as a positive control. The phosphorylated proteins were detected by autoradiography (top), and the proteins present in the assay are shown by immunoblotting (middle) or Ponceau staining (bottom). (D) Phos- phorylation of VRK1 by transfected Plk3. HEK293T cells were transfected with pCEFL-GST-Plk3 (5 g), and cell extracts were used for immunoprecipitation with monoclonal anti-Flag antibody (control) or monoclonal anti-GST antibody (Plk3). The immunoprecipitates were used for kinase assays using pGEX-GST-VRK1K179E as a substrate.

Techniques Used: Sequencing, In Vitro, Kinase Assay, Phospho-proteomics, Residue, Autoradiography, Staining, Positive Control, Western Blot, Transfection, Immunoprecipitation, Control

$FIG. 2. Expression, interaction, and colocalization of endogenous human VRK1 and Plk3 proteins. (A) The expression of endogenous VRK1 and Plk3 proteins in different cell lines was determined by immunoblotting. Cell lysate (20 g) from each cell line was fractionated in a sodium dodecyl sulfate-(10%) polyacrylamide gel and transferred to an Immobilon-P membrane. VRK1 was detected with the VC1 polyclonal antibody, and Plk3 was detected with a monoclonal antibody. (B) Interaction of endogenous VRK1 and Plk3 proteins in HEK293T cells. One milligram of cellular extract was used for immunoprecipitation of the endogenous VRK1 protein with a monoclonal antibody (1F6) or with a control antibody (monoclonal anti-Flag). The endogenous Plk3 immunoprecipitated was detected with a speciﬁc polyclonal antibody for Plk3. IP, immunoprecipi- tation; IB, immunoblot. (C). Colocalization in HEK293T cells. The endogenous VRK1 protein was detected with a speciﬁc polyclonal antibody (VE1). HEK293T cells were transfected with 3 g of Plk3 tagged with an HA epitope and detected with a mouse monoclonal antibody speciﬁc for the HA tag. Nuclei were identiﬁed by DAPI staining. (D). Colocalization in A549 cells. The endogenous VRK1 protein was detected with a speciﬁc monoclonal antibody (1F6). A549 cells were transfected with 3 g of Plk3 tagged with a Flag epitope and detected with a polyclonal antibody for the Flag epitope. Nuclei were stained with DAPI. Bar, 50 m.$
Figure Legend Snippet: FIG. 2. Expression, interaction, and colocalization of endogenous human VRK1 and Plk3 proteins. (A) The expression of endogenous VRK1 and Plk3 proteins in different cell lines was determined by immunoblotting. Cell lysate (20 g) from each cell line was fractionated in a sodium dodecyl sulfate-(10%) polyacrylamide gel and transferred to an Immobilon-P membrane. VRK1 was detected with the VC1 polyclonal antibody, and Plk3 was detected with a monoclonal antibody. (B) Interaction of endogenous VRK1 and Plk3 proteins in HEK293T cells. One milligram of cellular extract was used for immunoprecipitation of the endogenous VRK1 protein with a monoclonal antibody (1F6) or with a control antibody (monoclonal anti-Flag). The endogenous Plk3 immunoprecipitated was detected with a speciﬁc polyclonal antibody for Plk3. IP, immunoprecipi- tation; IB, immunoblot. (C). Colocalization in HEK293T cells. The endogenous VRK1 protein was detected with a speciﬁc polyclonal antibody (VE1). HEK293T cells were transfected with 3 g of Plk3 tagged with an HA epitope and detected with a mouse monoclonal antibody speciﬁc for the HA tag. Nuclei were identiﬁed by DAPI staining. (D). Colocalization in A549 cells. The endogenous VRK1 protein was detected with a speciﬁc monoclonal antibody (1F6). A549 cells were transfected with 3 g of Plk3 tagged with a Flag epitope and detected with a polyclonal antibody for the Flag epitope. Nuclei were stained with DAPI. Bar, 50 m.

Techniques Used: Expressing, Western Blot, Membrane, Immunoprecipitation, Control, Transfection, Staining, FLAG-tag

FIG. 3. Stable interaction between Plk3 and VRK1. (A) Reciprocal immunoprecipitation of transfected proteins. HEK293T cells were transfected with empty vector (pCEFL-HA) or plasmid pCEFL-HA-VRK1 (5 g) in combination with pCEFL-Flag-Plk3 (5 g). The cell lysate (top) was used for immunoprecipitation of proteins that bind to either VRK1 or Plk3 with an antibody speciﬁc for the corresponding epitope. (Bottom) Detection of the reciprocal proteins was determined by immunoblotting. (B) In vivo interaction of transfected VRK1 and Plk3 proteins. HEK293T cells were transfected with plasmids expressing active (pCEFL-GST-Plk3) and kinase-dead (pCEFL-GST-Plk3K52R) Plk3 in combina- tion with VRK1, either active (pCEFL-HA-VRK1), inactive (pCEFL-HA-VRK1K179E), or nonphosphorylatable by Plk3 (pCEFL-HA- VRK1S342A). Their correct expression was checked in cell lysates (bottom), which were used for a pull-down assay of proteins associated with the GST-Plk3 constructs. (C) Detection of the interaction between endogenous VRK1 and transfected Plk3. HEK293T cells were transfected with plasmid pCEFL-GST-Plk3 (6 g), pCEFL-GST-Plk3K52R (6 g), or pCEFL-GST (2 g) as a control. The expression of the proteins was determined by Western blotting (bottom). The different lysates were used for pull-down assay with glutathione-Sepharose to bring down the GST-Plk3 proteins. The pull-down proteins were detected with anti-VRK1 (1F6) monoclonal antibody. (D) Detection of the interaction between endogenous Plk3 and transfected VRK1. HEK293T cells were transfected with plasmid pCEFL-GST-VRK1 (8 g), pCEFL-GST-VRK1K179E (8 g), or pCEFL-GST (2 g) as a control. The expression of the proteins was determined by Western blotting (bottom). The different lysates were used for a pull-down assay with glutathione-Sepharose to bring down the GST-VRK1 proteins. In the pull-down assay, the endogenous Plk3 protein was detected with a speciﬁc antibody.

Figure Legend Snippet: FIG. 3. Stable interaction between Plk3 and VRK1. (A) Reciprocal immunoprecipitation of transfected proteins. HEK293T cells were transfected with empty vector (pCEFL-HA) or plasmid pCEFL-HA-VRK1 (5 g) in combination with pCEFL-Flag-Plk3 (5 g). The cell lysate (top) was used for immunoprecipitation of proteins that bind to either VRK1 or Plk3 with an antibody speciﬁc for the corresponding epitope. (Bottom) Detection of the reciprocal proteins was determined by immunoblotting. (B) In vivo interaction of transfected VRK1 and Plk3 proteins. HEK293T cells were transfected with plasmids expressing active (pCEFL-GST-Plk3) and kinase-dead (pCEFL-GST-Plk3K52R) Plk3 in combina- tion with VRK1, either active (pCEFL-HA-VRK1), inactive (pCEFL-HA-VRK1K179E), or nonphosphorylatable by Plk3 (pCEFL-HA- VRK1S342A). Their correct expression was checked in cell lysates (bottom), which were used for a pull-down assay of proteins associated with the GST-Plk3 constructs. (C) Detection of the interaction between endogenous VRK1 and transfected Plk3. HEK293T cells were transfected with plasmid pCEFL-GST-Plk3 (6 g), pCEFL-GST-Plk3K52R (6 g), or pCEFL-GST (2 g) as a control. The expression of the proteins was determined by Western blotting (bottom). The different lysates were used for pull-down assay with glutathione-Sepharose to bring down the GST-Plk3 proteins. The pull-down proteins were detected with anti-VRK1 (1F6) monoclonal antibody. (D) Detection of the interaction between endogenous Plk3 and transfected VRK1. HEK293T cells were transfected with plasmid pCEFL-GST-VRK1 (8 g), pCEFL-GST-VRK1K179E (8 g), or pCEFL-GST (2 g) as a control. The expression of the proteins was determined by Western blotting (bottom). The different lysates were used for a pull-down assay with glutathione-Sepharose to bring down the GST-VRK1 proteins. In the pull-down assay, the endogenous Plk3 protein was detected with a speciﬁc antibody.

Techniques Used: Immunoprecipitation, Transfection, Plasmid Preparation, Western Blot, In Vivo, Expressing, Pull Down Assay, Construct, Control

FIG. 4. Mapping of the region of VRK1 interacting with Plk3. (A) HEK293T cells were transfected with plasmids expressing mammalian GST-VRK1 (FL, full-length; C, C-terminal region) and Plk3 tagged with a Flag epitope. HEK293T cells were cotransfected with constructs of VRK1-FL (8 g) and VRK1-C (6 g) fused to GST and with Flag-Plk3 (3 g). The correct expression levels of proteins were checked in the lysates. The lysates were used for a pull-down assay with glutathione-Sepharose beads, and the associated proteins were detected in Western blots (top). VRK1 constructs were detected with an anti-GST antibody. Plk3 was detected with an anti-Flag antibody. (B) HEK293T cells were transfected with plasmids expressing different regions of human VRK1 tagged with myc and a mammalian construct expressing GST-Plk3. HEK293T cells were cotransfected with constructs of VRK1 fused to myc expressing different regions of the VRK1 protein (pCDNA-myc-VRK1-FL [5 g], pCDNA- myc-VRK1-NL [5 g], and pCDNA-myc-VRK1-NS [9 g]) and with Plk3 (6 g) fused to GST. The lysates were used for a pull-down assay with glutathione-Sepharose beads, and the associated proteins were detected in Western blots (top). GST empty vector was used as a control. Plk3 was detected with an anti-GST antibody. VRK1 was detected with a speciﬁc rabbit polyclonal antibody (VE1). (C) Potential region of interaction between VRK1 and Plk3.

Figure Legend Snippet: FIG. 4. Mapping of the region of VRK1 interacting with Plk3. (A) HEK293T cells were transfected with plasmids expressing mammalian GST-VRK1 (FL, full-length; C, C-terminal region) and Plk3 tagged with a Flag epitope. HEK293T cells were cotransfected with constructs of VRK1-FL (8 g) and VRK1-C (6 g) fused to GST and with Flag-Plk3 (3 g). The correct expression levels of proteins were checked in the lysates. The lysates were used for a pull-down assay with glutathione-Sepharose beads, and the associated proteins were detected in Western blots (top). VRK1 constructs were detected with an anti-GST antibody. Plk3 was detected with an anti-Flag antibody. (B) HEK293T cells were transfected with plasmids expressing different regions of human VRK1 tagged with myc and a mammalian construct expressing GST-Plk3. HEK293T cells were cotransfected with constructs of VRK1 fused to myc expressing different regions of the VRK1 protein (pCDNA-myc-VRK1-FL [5 g], pCDNA- myc-VRK1-NL [5 g], and pCDNA-myc-VRK1-NS [9 g]) and with Plk3 (6 g) fused to GST. The lysates were used for a pull-down assay with glutathione-Sepharose beads, and the associated proteins were detected in Western blots (top). GST empty vector was used as a control. Plk3 was detected with an anti-GST antibody. VRK1 was detected with a speciﬁc rabbit polyclonal antibody (VE1). (C) Potential region of interaction between VRK1 and Plk3.

Techniques Used: Transfection, Expressing, FLAG-tag, Construct, Pull Down Assay, Western Blot, Plasmid Preparation, Control

FIG. 7. Effect of VRK1 on MEK1-Plk3 fragmentation pathway phenotype. (A) Effects on Golgi fragmentation induced by MEK1. At the bottom is shown the quantiﬁcation of the effect. (B) Effects on Golgi fragmentation induced by Plk3. HeLa cells were transfected with constructs expressing the indicated proteins. The MEK1 protein is constitutively active. The transfected cells were identiﬁed by the use of green ﬂuorescent protein (GFP) as a ﬂuorescent tracer protein or of an antibody against the tag in the transfected protein. The Golgi apparatus was detected with a polyclonal antibody speciﬁc for giantin. Bar, 20 m. TC, transfection control with empty vector pEGFP-N1. The quantiﬁcation of the effects in panels A and B are shown at the bottom of the corresponding column. At least 100 cells from three independent experiments were counted for each type of transfection, and the proportion of cells with fragmented Golgi complexes was determined. In the left column, the reference value is the fragmentation induced by MEK1 (b). In the right column, the reference value is the fragmentation induced by Plk3 (a). The mean values with their standard deviations are represented in the bar graph. *, P 0.05; **, P 0.005.

Figure Legend Snippet: FIG. 7. Effect of VRK1 on MEK1-Plk3 fragmentation pathway phenotype. (A) Effects on Golgi fragmentation induced by MEK1. At the bottom is shown the quantiﬁcation of the effect. (B) Effects on Golgi fragmentation induced by Plk3. HeLa cells were transfected with constructs expressing the indicated proteins. The MEK1 protein is constitutively active. The transfected cells were identiﬁed by the use of green ﬂuorescent protein (GFP) as a ﬂuorescent tracer protein or of an antibody against the tag in the transfected protein. The Golgi apparatus was detected with a polyclonal antibody speciﬁc for giantin. Bar, 20 m. TC, transfection control with empty vector pEGFP-N1. The quantiﬁcation of the effects in panels A and B are shown at the bottom of the corresponding column. At least 100 cells from three independent experiments were counted for each type of transfection, and the proportion of cells with fragmented Golgi complexes was determined. In the left column, the reference value is the fragmentation induced by MEK1 (b). In the right column, the reference value is the fragmentation induced by Plk3 (a). The mean values with their standard deviations are represented in the bar graph. *, P 0.05; **, P 0.005.

Techniques Used: Transfection, Construct, Expressing, Control, Plasmid Preparation

FIG. 8. Knockdown of VRK1 blocks the Golgi fragmentation phenotype induced by either MEK1 or Plk3. (A) Levels of VRK1 endogenous protein in nontransfected cells or cells transfected with siRNA control or with siRNA speciﬁc for VRK1 were determined after 5 days. NTC, nontransfected cells. (B) Photograph of HeLa cells transfected with siControl (left) or siVRK1 (right) for 5 days. (C) Immunoﬂuorescence of endogenous VRK1 protein detected with VC polyclonal antibody. The Golgi marker GM130 was used to detect the fragmentation of the Golgi apparatus. Cells were treated with the indicated siControl or speciﬁc siVRK1 as well as with MEK1 or Plk3 as an inducer of Golgi fragmentation, as indicated in each row. Five days after siRNA treatment, cells were retransfected with 3 g of constitutively active MEK1(S218/222E, 32–51) or 3 g of Plk3. Fifteen or 13 h after retransfection, cells were ﬁxed and stained with polyclonal VC1 antibody to VRK1 (green) and a speciﬁc monoclonal antibody to GM130 (red). Bar, 50 m. Transfected cells were identiﬁed by GFP or by an anti-tag antibody. The number of intracellular particles (objects) in transfected cells was analyzed by counting them using the ImageJ program (http://rsb.info.nih.gov/ij; developed by Wayne Rasband, National Institutes of Health, Bethesda, MD). The mean values with their standard deviations are shown in the bar graph. A minimum of 20 cells were counted in each case.

Figure Legend Snippet: FIG. 8. Knockdown of VRK1 blocks the Golgi fragmentation phenotype induced by either MEK1 or Plk3. (A) Levels of VRK1 endogenous protein in nontransfected cells or cells transfected with siRNA control or with siRNA speciﬁc for VRK1 were determined after 5 days. NTC, nontransfected cells. (B) Photograph of HeLa cells transfected with siControl (left) or siVRK1 (right) for 5 days. (C) Immunoﬂuorescence of endogenous VRK1 protein detected with VC polyclonal antibody. The Golgi marker GM130 was used to detect the fragmentation of the Golgi apparatus. Cells were treated with the indicated siControl or speciﬁc siVRK1 as well as with MEK1 or Plk3 as an inducer of Golgi fragmentation, as indicated in each row. Five days after siRNA treatment, cells were retransfected with 3 g of constitutively active MEK1(S218/222E, 32–51) or 3 g of Plk3. Fifteen or 13 h after retransfection, cells were ﬁxed and stained with polyclonal VC1 antibody to VRK1 (green) and a speciﬁc monoclonal antibody to GM130 (red). Bar, 50 m. Transfected cells were identiﬁed by GFP or by an anti-tag antibody. The number of intracellular particles (objects) in transfected cells was analyzed by counting them using the ImageJ program (http://rsb.info.nih.gov/ij; developed by Wayne Rasband, National Institutes of Health, Bethesda, MD). The mean values with their standard deviations are shown in the bar graph. A minimum of 20 cells were counted in each case.

Techniques Used: Knockdown, Transfection, Control, Marker, Staining

Image Search Results

The most sensitive new pS129 α-synuclein assay (left) was characterized using wild-type (WT) mouse brain tissue extracts and PLK3-transfected HEK293 cell lysates ( a , b ), while testing of the most sensitive total α-synuclein assay (right) utilized WT mouse brain tissue extracts and WT HEK293 cell lysates ( c , d ). Extracts and lysates were serially diluted using assay buffer. Associated assay data for all formulations tested are contained in Supplementary Table . Inter-assay variation (RSD) data presented in ( b ) and ( d ) were calculated from three individual dilution curves constructed and measured on separate days, with data only shown for extract/lysate dilutions above the lower limit of quantification (LLoQ). Phospho-S129 ( e , f ) and total ( g , h ) assay specificity were also assessed using SNCA knock-out (KO) HEK293 cell lysates and Snca KO mouse brain tissue extracts. Protein concentrations in each sample prior to serial dilution were as follows: WT mouse brain supernatant, 4.93 mg/mL; KO mouse brain supernatant, 5.48 mg/mL; WT HEK293 supernatant, 4.77 mg/mL; KO HEK293 supernatant, 5.6 mg/mL; PLK3-HEK293 supernatant, 6.55 mg/mL. The limit of detection (LoD) and LLoQ were defined as described in Fig. . Data in ( a , b , g , h ) represent mean ± standard deviation. For all standard curves; number of independent experiments ( N ) = 3, number of replicates in each experiment ( n = 3). AU arbitrary units.

Journal: NPJ Parkinson's Disease

Article Title: Novel tools to quantify total, phospho-Ser129 and aggregated alpha-synuclein in the mouse brain

doi: 10.1038/s41531-024-00830-y

Figure Lengend Snippet: The most sensitive new pS129 α-synuclein assay (left) was characterized using wild-type (WT) mouse brain tissue extracts and PLK3-transfected HEK293 cell lysates ( a , b ), while testing of the most sensitive total α-synuclein assay (right) utilized WT mouse brain tissue extracts and WT HEK293 cell lysates ( c , d ). Extracts and lysates were serially diluted using assay buffer. Associated assay data for all formulations tested are contained in Supplementary Table . Inter-assay variation (RSD) data presented in ( b ) and ( d ) were calculated from three individual dilution curves constructed and measured on separate days, with data only shown for extract/lysate dilutions above the lower limit of quantification (LLoQ). Phospho-S129 ( e , f ) and total ( g , h ) assay specificity were also assessed using SNCA knock-out (KO) HEK293 cell lysates and Snca KO mouse brain tissue extracts. Protein concentrations in each sample prior to serial dilution were as follows: WT mouse brain supernatant, 4.93 mg/mL; KO mouse brain supernatant, 5.48 mg/mL; WT HEK293 supernatant, 4.77 mg/mL; KO HEK293 supernatant, 5.6 mg/mL; PLK3-HEK293 supernatant, 6.55 mg/mL. The limit of detection (LoD) and LLoQ were defined as described in Fig. . Data in ( a , b , g , h ) represent mean ± standard deviation. For all standard curves; number of independent experiments ( N ) = 3, number of replicates in each experiment ( n = 3). AU arbitrary units.

Article Snippet: Phosphorylation was induced by incubating 1 μg of recombinant human PLK3 protein (Thermo Fisher Scientific, MA, USA; #PR7316B) with 50 μL of mouse WT α-synuclein in kinase reaction buffer for 8 h at 30 °C.

Techniques: Transfection, Inter Assay, Construct, Knock-Out, Serial Dilution, Standard Deviation

Journal: Molecular and Cellular Biology

Article Title: Plk3 Interacts with and Specifically Phosphorylates VRK1 in Ser 342 , a Downstream Target in a Pathway That Induces Golgi Fragmentation

doi: 10.1128/mcb.01341-08

Figure Lengend Snippet: FIG. 1. VRK1 is phosphorylated in Ser342 by Plk3. (A) Sequence in the C-terminal region of VRK1 containing a conserved sequence that is a potential target for Plks. (B) In vitro kinase assay to determine the phosphorylation order between Plk3 and VRK1 and the residue phosphorylated. Plk3 and human VRK1 were expressed in E. coli and puriﬁed for the in vitro kinase assay. KE, K179E substitution in VRK1 that is kinase dead. The phosphorylated proteins were detected by autoradiography (top), and the proteins present in the assay are shown by Coomassie blue staining (bottom). (C) In vitro kinase assay to demonstrate that VRK1 does not phosphorylate kinase-dead Plk3. pGEX-GST-VRK1, pGEX-GST-p53 (1-82), and pGEX-GST-Plk3K52R were expressed in E. coli. The assay was carried out with a constant amount of GST-VRK1 and increasing amounts (as indicated) of GST-Plk3. GST-p53 was used as a positive control. The phosphorylated proteins were detected by autoradiography (top), and the proteins present in the assay are shown by immunoblotting (middle) or Ponceau staining (bottom). (D) Phos- phorylation of VRK1 by transfected Plk3. HEK293T cells were transfected with pCEFL-GST-Plk3 (5 g), and cell extracts were used for immunoprecipitation with monoclonal anti-Flag antibody (control) or monoclonal anti-GST antibody (Plk3). The immunoprecipitates were used for kinase assays using pGEX-GST-VRK1K179E as a substrate.

Article Snippet: Human Plk3 protein was detected with a monoclonal antibody (clone B37-2) (BD Pharmingen, San Diego, CA) or a polyclonal antibody (10977-1AP) (ProteinTech Group Inc., Chicago, IL).

Techniques: Sequencing, In Vitro, Kinase Assay, Phospho-proteomics, Residue, Autoradiography, Staining, Positive Control, Western Blot, Transfection, Immunoprecipitation, Control

Journal: Molecular and Cellular Biology

Article Title: Plk3 Interacts with and Specifically Phosphorylates VRK1 in Ser 342 , a Downstream Target in a Pathway That Induces Golgi Fragmentation

doi: 10.1128/mcb.01341-08

Figure Lengend Snippet: FIG. 2. Expression, interaction, and colocalization of endogenous human VRK1 and Plk3 proteins. (A) The expression of endogenous VRK1 and Plk3 proteins in different cell lines was determined by immunoblotting. Cell lysate (20 g) from each cell line was fractionated in a sodium dodecyl sulfate-(10%) polyacrylamide gel and transferred to an Immobilon-P membrane. VRK1 was detected with the VC1 polyclonal antibody, and Plk3 was detected with a monoclonal antibody. (B) Interaction of endogenous VRK1 and Plk3 proteins in HEK293T cells. One milligram of cellular extract was used for immunoprecipitation of the endogenous VRK1 protein with a monoclonal antibody (1F6) or with a control antibody (monoclonal anti-Flag). The endogenous Plk3 immunoprecipitated was detected with a speciﬁc polyclonal antibody for Plk3. IP, immunoprecipi- tation; IB, immunoblot. (C). Colocalization in HEK293T cells. The endogenous VRK1 protein was detected with a speciﬁc polyclonal antibody (VE1). HEK293T cells were transfected with 3 g of Plk3 tagged with an HA epitope and detected with a mouse monoclonal antibody speciﬁc for the HA tag. Nuclei were identiﬁed by DAPI staining. (D). Colocalization in A549 cells. The endogenous VRK1 protein was detected with a speciﬁc monoclonal antibody (1F6). A549 cells were transfected with 3 g of Plk3 tagged with a Flag epitope and detected with a polyclonal antibody for the Flag epitope. Nuclei were stained with DAPI. Bar, 50 m.

Techniques: Expressing, Western Blot, Membrane, Immunoprecipitation, Control, Transfection, Staining, FLAG-tag

Journal: Molecular and Cellular Biology

Article Title: Plk3 Interacts with and Specifically Phosphorylates VRK1 in Ser 342 , a Downstream Target in a Pathway That Induces Golgi Fragmentation

doi: 10.1128/mcb.01341-08

Figure Lengend Snippet: FIG. 3. Stable interaction between Plk3 and VRK1. (A) Reciprocal immunoprecipitation of transfected proteins. HEK293T cells were transfected with empty vector (pCEFL-HA) or plasmid pCEFL-HA-VRK1 (5 g) in combination with pCEFL-Flag-Plk3 (5 g). The cell lysate (top) was used for immunoprecipitation of proteins that bind to either VRK1 or Plk3 with an antibody speciﬁc for the corresponding epitope. (Bottom) Detection of the reciprocal proteins was determined by immunoblotting. (B) In vivo interaction of transfected VRK1 and Plk3 proteins. HEK293T cells were transfected with plasmids expressing active (pCEFL-GST-Plk3) and kinase-dead (pCEFL-GST-Plk3K52R) Plk3 in combina- tion with VRK1, either active (pCEFL-HA-VRK1), inactive (pCEFL-HA-VRK1K179E), or nonphosphorylatable by Plk3 (pCEFL-HA- VRK1S342A). Their correct expression was checked in cell lysates (bottom), which were used for a pull-down assay of proteins associated with the GST-Plk3 constructs. (C) Detection of the interaction between endogenous VRK1 and transfected Plk3. HEK293T cells were transfected with plasmid pCEFL-GST-Plk3 (6 g), pCEFL-GST-Plk3K52R (6 g), or pCEFL-GST (2 g) as a control. The expression of the proteins was determined by Western blotting (bottom). The different lysates were used for pull-down assay with glutathione-Sepharose to bring down the GST-Plk3 proteins. The pull-down proteins were detected with anti-VRK1 (1F6) monoclonal antibody. (D) Detection of the interaction between endogenous Plk3 and transfected VRK1. HEK293T cells were transfected with plasmid pCEFL-GST-VRK1 (8 g), pCEFL-GST-VRK1K179E (8 g), or pCEFL-GST (2 g) as a control. The expression of the proteins was determined by Western blotting (bottom). The different lysates were used for a pull-down assay with glutathione-Sepharose to bring down the GST-VRK1 proteins. In the pull-down assay, the endogenous Plk3 protein was detected with a speciﬁc antibody.

Techniques: Immunoprecipitation, Transfection, Plasmid Preparation, Western Blot, In Vivo, Expressing, Pull Down Assay, Construct, Control

Journal: Molecular and Cellular Biology

Article Title: Plk3 Interacts with and Specifically Phosphorylates VRK1 in Ser 342 , a Downstream Target in a Pathway That Induces Golgi Fragmentation

doi: 10.1128/mcb.01341-08

Figure Lengend Snippet: FIG. 4. Mapping of the region of VRK1 interacting with Plk3. (A) HEK293T cells were transfected with plasmids expressing mammalian GST-VRK1 (FL, full-length; C, C-terminal region) and Plk3 tagged with a Flag epitope. HEK293T cells were cotransfected with constructs of VRK1-FL (8 g) and VRK1-C (6 g) fused to GST and with Flag-Plk3 (3 g). The correct expression levels of proteins were checked in the lysates. The lysates were used for a pull-down assay with glutathione-Sepharose beads, and the associated proteins were detected in Western blots (top). VRK1 constructs were detected with an anti-GST antibody. Plk3 was detected with an anti-Flag antibody. (B) HEK293T cells were transfected with plasmids expressing different regions of human VRK1 tagged with myc and a mammalian construct expressing GST-Plk3. HEK293T cells were cotransfected with constructs of VRK1 fused to myc expressing different regions of the VRK1 protein (pCDNA-myc-VRK1-FL [5 g], pCDNA- myc-VRK1-NL [5 g], and pCDNA-myc-VRK1-NS [9 g]) and with Plk3 (6 g) fused to GST. The lysates were used for a pull-down assay with glutathione-Sepharose beads, and the associated proteins were detected in Western blots (top). GST empty vector was used as a control. Plk3 was detected with an anti-GST antibody. VRK1 was detected with a speciﬁc rabbit polyclonal antibody (VE1). (C) Potential region of interaction between VRK1 and Plk3.

Techniques: Transfection, Expressing, FLAG-tag, Construct, Pull Down Assay, Western Blot, Plasmid Preparation, Control

Journal: Molecular and Cellular Biology

Article Title: Plk3 Interacts with and Specifically Phosphorylates VRK1 in Ser 342 , a Downstream Target in a Pathway That Induces Golgi Fragmentation

doi: 10.1128/mcb.01341-08

Figure Lengend Snippet: FIG. 7. Effect of VRK1 on MEK1-Plk3 fragmentation pathway phenotype. (A) Effects on Golgi fragmentation induced by MEK1. At the bottom is shown the quantiﬁcation of the effect. (B) Effects on Golgi fragmentation induced by Plk3. HeLa cells were transfected with constructs expressing the indicated proteins. The MEK1 protein is constitutively active. The transfected cells were identiﬁed by the use of green ﬂuorescent protein (GFP) as a ﬂuorescent tracer protein or of an antibody against the tag in the transfected protein. The Golgi apparatus was detected with a polyclonal antibody speciﬁc for giantin. Bar, 20 m. TC, transfection control with empty vector pEGFP-N1. The quantiﬁcation of the effects in panels A and B are shown at the bottom of the corresponding column. At least 100 cells from three independent experiments were counted for each type of transfection, and the proportion of cells with fragmented Golgi complexes was determined. In the left column, the reference value is the fragmentation induced by MEK1 (b). In the right column, the reference value is the fragmentation induced by Plk3 (a). The mean values with their standard deviations are represented in the bar graph. *, P 0.05; **, P 0.005.

Techniques: Transfection, Construct, Expressing, Control, Plasmid Preparation

Journal: Molecular and Cellular Biology

Article Title: Plk3 Interacts with and Specifically Phosphorylates VRK1 in Ser 342 , a Downstream Target in a Pathway That Induces Golgi Fragmentation

doi: 10.1128/mcb.01341-08

Figure Lengend Snippet: FIG. 8. Knockdown of VRK1 blocks the Golgi fragmentation phenotype induced by either MEK1 or Plk3. (A) Levels of VRK1 endogenous protein in nontransfected cells or cells transfected with siRNA control or with siRNA speciﬁc for VRK1 were determined after 5 days. NTC, nontransfected cells. (B) Photograph of HeLa cells transfected with siControl (left) or siVRK1 (right) for 5 days. (C) Immunoﬂuorescence of endogenous VRK1 protein detected with VC polyclonal antibody. The Golgi marker GM130 was used to detect the fragmentation of the Golgi apparatus. Cells were treated with the indicated siControl or speciﬁc siVRK1 as well as with MEK1 or Plk3 as an inducer of Golgi fragmentation, as indicated in each row. Five days after siRNA treatment, cells were retransfected with 3 g of constitutively active MEK1(S218/222E, 32–51) or 3 g of Plk3. Fifteen or 13 h after retransfection, cells were ﬁxed and stained with polyclonal VC1 antibody to VRK1 (green) and a speciﬁc monoclonal antibody to GM130 (red). Bar, 50 m. Transfected cells were identiﬁed by GFP or by an anti-tag antibody. The number of intracellular particles (objects) in transfected cells was analyzed by counting them using the ImageJ program (http://rsb.info.nih.gov/ij; developed by Wayne Rasband, National Institutes of Health, Bethesda, MD). The mean values with their standard deviations are shown in the bar graph. A minimum of 20 cells were counted in each case.

Techniques: Knockdown, Transfection, Control, Marker, Staining

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