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Tocris ck1 inhibitor pf 670462
Ck1 Inhibitor Pf 670462, supplied by Tocris, used in various techniques. Bioz Stars score: 94/100, based on 36 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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Average 94 stars, based on 36 article reviews

ck1 inhibitor pf 670462 - by Bioz Stars, 2026-07

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Fig. 3 Wnt pathway activation induces γ-secretase activity stimulating EpICD production and its subsequent nuclear translocation. a γ-secretase activity quantified in cell lines treated with either ctrl-His or Wnt3A (100 ng/mL) or EpEX-His (250 ng/mL) for 4 h. Western blot analysis showing phosphorylated presenilin-2 (PS2) in (b) indicated cells treated with ctrl-His (100 ng/mL), Wnt3A (100 ng/mL) or EpEX-His (250 ng/mL) for 20 min or with 1 h pre-treated (c) BIO (50 µM) or (d) PF-670462 (50 µM) in HCT116 cells. e Western blot analysis showing overall EpICD level in total lysate, and f representative IFS images (Scale bar: 10 μm) showing EpICD localization in HCT116 cells treated with either ctrl-His or Wnt3A (100 ng/mL) or EpEX-His (250 ng/mL) for 8 h. g Quantification of nuclear EpICD levels from 20 different cells per group. h Illustration showing the proposed mechanism of PS2 phosphorylation by GSK3β and CK1 to activate γ-secretase, which then cleaves EpICD from intact full-length EpCAM. i-j γ-Secretase activity and k, l Western blot analysis showing phosphorylation of PS2 in indicated cell lines and PDOs treated with either ctrl-IgG or hEpAb2-6 (20 μg/mL) for 4 h. Data were analyzed using (a, g) one-way ANOVA followed by Tukey’s test for multiple comparisons and i, j two-tailed t test. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. Ctrl: control

Journal: Journal of biomedical science

Article Title: Intracellular domain of epithelial cell adhesion molecule induces Wnt receptor transcription to promote colorectal cancer progression.

doi: 10.1186/s12929-024-01057-y

Figure Lengend Snippet: Fig. 3 Wnt pathway activation induces γ-secretase activity stimulating EpICD production and its subsequent nuclear translocation. a γ-secretase activity quantified in cell lines treated with either ctrl-His or Wnt3A (100 ng/mL) or EpEX-His (250 ng/mL) for 4 h. Western blot analysis showing phosphorylated presenilin-2 (PS2) in (b) indicated cells treated with ctrl-His (100 ng/mL), Wnt3A (100 ng/mL) or EpEX-His (250 ng/mL) for 20 min or with 1 h pre-treated (c) BIO (50 µM) or (d) PF-670462 (50 µM) in HCT116 cells. e Western blot analysis showing overall EpICD level in total lysate, and f representative IFS images (Scale bar: 10 μm) showing EpICD localization in HCT116 cells treated with either ctrl-His or Wnt3A (100 ng/mL) or EpEX-His (250 ng/mL) for 8 h. g Quantification of nuclear EpICD levels from 20 different cells per group. h Illustration showing the proposed mechanism of PS2 phosphorylation by GSK3β and CK1 to activate γ-secretase, which then cleaves EpICD from intact full-length EpCAM. i-j γ-Secretase activity and k, l Western blot analysis showing phosphorylation of PS2 in indicated cell lines and PDOs treated with either ctrl-IgG or hEpAb2-6 (20 μg/mL) for 4 h. Data were analyzed using (a, g) one-way ANOVA followed by Tukey’s test for multiple comparisons and i, j two-tailed t test. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. Ctrl: control

Article Snippet: To assess the levels of GSK3 and CK1 kinase activities, 106 cells were seeded overnight and treated with GSK3 inhibitor BIO (Sigma) or CK1 inhibitor PF-670462 (selleckchem) for 8 h. Cells were then lysed with RIPA buffer and subjected to western blot analysis for phosphorylated Presenilin2.

Techniques: Activation Assay, Activity Assay, Translocation Assay, Western Blot, Phospho-proteomics, Two Tailed Test, Control

Inhibition of WNT signaling blocks the effect of WNT5A. A) Scheme of action and outcomes of CK1 and CK1 inhibitors in the WNT pathway. B) Representative WBs from cells used in C-E). Both CK1 inhibitors (CK1 inh. I: 5 μM PF670462, CK1 inh. II: 5 μM D4476) are able to effectively block WNT signaling induced by WNT5A, witnessed by diminishment of PS-DVL2 and PS-DVL3 (black triangle). Quantification of WBs is available in Supplementary G-H. C-E) Cells were pretreated with 0.1 μM LGK974 (inhibitor of Porcupine) and then with either control or WNT5A CM for 18 h. The two conditions treated with WNT5A also contained CK1 inhibitors during the whole WNT5A CM treatment. Then the cells were used for functional analyses. C) Activation of WNT/PCP pathway by WNT5A increases self-renewal potential of single cells in sphere forming assay. WNT5A induces migration potential ( D ) as well as the invasion capacity of HGSC cells in a transwell assay ( E ) and this effect can be reduced or completely blocked by inhibitors of CK1. Results are expressed as sphere, migration or invasion index, which is a value normalized to mean of the triplicate control condition in each experiment. Representative images of transwell membranes are shown on the right side of corresponding graph, scale bar = 100 μm. F-K) Genetic ablation of WNT pathway diminished the effect of WNT5A in functional assays. DVL3, a critical component of WNT pathway, was inactivated in Kuramochi cells using the CRISPR/Cas9 system. F) Targeting strategy and sequencing results of individual DVL3 KO clones. Frame shift mutation in exon 4 was introduced into each allele, thus generating homozygous knock-out cell lines. G) WB analysis of DVL3 and DVL2 in wild-type (WT) and two DVL3 KO clones (A7 and C2) used in functional assays in H-K, β-ACTIN served as a loading control. H) DVL3 KO cells have reduced the ability to form spheres in comparison to WT cells. I) WNT5A increased the self-renewal potential of single cells in a sphere forming assay on WT cells but not in DVL3 KO clones. Similarly, WNT5A promotes migration ( J ) and invasion ( K ) in WT cells but not in DVL3 KO cells. Data information: In (C-E, H-K), data is presented as mean ± SD. * = P ≤ 0.05; ** = P ≤ 0.01; *** = P ≤ 0.001. **** = P < 0.0001 (Tukey post-hoc test of one-way ANOVA), n.s. = non-significant ( P > 0.05). Experiments were done in triplicate, N = 3.

Journal: Theranostics

Article Title: WNT signaling inducing activity in ascites predicts poor outcome in ovarian cancer

doi: 10.7150/thno.37423

Figure Lengend Snippet: Inhibition of WNT signaling blocks the effect of WNT5A. A) Scheme of action and outcomes of CK1 and CK1 inhibitors in the WNT pathway. B) Representative WBs from cells used in C-E). Both CK1 inhibitors (CK1 inh. I: 5 μM PF670462, CK1 inh. II: 5 μM D4476) are able to effectively block WNT signaling induced by WNT5A, witnessed by diminishment of PS-DVL2 and PS-DVL3 (black triangle). Quantification of WBs is available in Supplementary G-H. C-E) Cells were pretreated with 0.1 μM LGK974 (inhibitor of Porcupine) and then with either control or WNT5A CM for 18 h. The two conditions treated with WNT5A also contained CK1 inhibitors during the whole WNT5A CM treatment. Then the cells were used for functional analyses. C) Activation of WNT/PCP pathway by WNT5A increases self-renewal potential of single cells in sphere forming assay. WNT5A induces migration potential ( D ) as well as the invasion capacity of HGSC cells in a transwell assay ( E ) and this effect can be reduced or completely blocked by inhibitors of CK1. Results are expressed as sphere, migration or invasion index, which is a value normalized to mean of the triplicate control condition in each experiment. Representative images of transwell membranes are shown on the right side of corresponding graph, scale bar = 100 μm. F-K) Genetic ablation of WNT pathway diminished the effect of WNT5A in functional assays. DVL3, a critical component of WNT pathway, was inactivated in Kuramochi cells using the CRISPR/Cas9 system. F) Targeting strategy and sequencing results of individual DVL3 KO clones. Frame shift mutation in exon 4 was introduced into each allele, thus generating homozygous knock-out cell lines. G) WB analysis of DVL3 and DVL2 in wild-type (WT) and two DVL3 KO clones (A7 and C2) used in functional assays in H-K, β-ACTIN served as a loading control. H) DVL3 KO cells have reduced the ability to form spheres in comparison to WT cells. I) WNT5A increased the self-renewal potential of single cells in a sphere forming assay on WT cells but not in DVL3 KO clones. Similarly, WNT5A promotes migration ( J ) and invasion ( K ) in WT cells but not in DVL3 KO cells. Data information: In (C-E, H-K), data is presented as mean ± SD. * = P ≤ 0.05; ** = P ≤ 0.01; *** = P ≤ 0.001. **** = P < 0.0001 (Tukey post-hoc test of one-way ANOVA), n.s. = non-significant ( P > 0.05). Experiments were done in triplicate, N = 3.

Article Snippet: The Porcupine inhibitor LGK974 (974-02; Stem RD) was used at a 0.1 μM concentration while CK1 inhibitors PF670462 [sc-204180A; Santa Cruz Biotechnology (SCBT)] and D4476 (218696; Calbiochem) were used in 5 μM final concentration.

Techniques: Inhibition, Blocking Assay, Control, Functional Assay, Activation Assay, Migration, Transwell Assay, CRISPR, Sequencing, Clone Assay, Mutagenesis, Knock-Out, Comparison

Figure 1 Analysis of B lymphocyte (MEC1) cell migration by live cell imaging. (A) A photomicrograph showing strongly polarized migrating MEC1 cells with the clearly defined leading and trailing edge. Arrows indicate the direction of migration. Size bar = 10 μm. (B) Snapshots of migrating, cell tracker-stained, MEC1 cells from time-lapse microscopy at approx. time: 0, 5, 10 and 15 min. Individual moving cells are indicated by the same color-coded arrow in each snapshot. Size bar = 10 μm. (C) MEC1 cells were seeded on glass-bottom plates coated with human plasma fibronectin. Control condition was captured first, for 30 min. Subsequently, CCL19 chemokine was added to the cells and the same position was scanned for additional 30 min. Last, cells were inhibited with PF670462 and followed another 30 min. (D) Four-compartment glass bottom plates were used for parallel tracking the cells (control [1], CCL19-treated [2] and CCL19/CK1 inhibitor-treated [3]). (E) Ibidi chamber for self-insertion was inserted in a glass-bottom plate, coated with Fibronectin and cell-tracker stained MEC1 cells were seeded. Ibidi chambers allow parallel scanning in two conditions (control [1a] and CCL19-stimulated [2a] first, captured for 30 min). After that, cells were stimulated with the chemokine [1b] and one of the pools was inhibited with the CK1 inh I [2b] and scanned for additional 30 min. From the first period, MEC1 migration in control and CCL19- stimulated wells [1a, 2a] is compared to each other and after that, CCL19 only and CCL19 + CK1 inh [1b, 2b] are compared to each other. At least 50 cells was tracked in each condition. ***, P < 0.001 (Kruskal-Wallis test).

Journal: Cell communication and signaling : CCS

Article Title: Asymmetry of VANGL2 in migrating lymphocytes as a tool to monitor activity of the mammalian WNT/planar cell polarity pathway.

doi: 10.1186/s12964-014-0079-1

Figure Lengend Snippet: Figure 1 Analysis of B lymphocyte (MEC1) cell migration by live cell imaging. (A) A photomicrograph showing strongly polarized migrating MEC1 cells with the clearly defined leading and trailing edge. Arrows indicate the direction of migration. Size bar = 10 μm. (B) Snapshots of migrating, cell tracker-stained, MEC1 cells from time-lapse microscopy at approx. time: 0, 5, 10 and 15 min. Individual moving cells are indicated by the same color-coded arrow in each snapshot. Size bar = 10 μm. (C) MEC1 cells were seeded on glass-bottom plates coated with human plasma fibronectin. Control condition was captured first, for 30 min. Subsequently, CCL19 chemokine was added to the cells and the same position was scanned for additional 30 min. Last, cells were inhibited with PF670462 and followed another 30 min. (D) Four-compartment glass bottom plates were used for parallel tracking the cells (control [1], CCL19-treated [2] and CCL19/CK1 inhibitor-treated [3]). (E) Ibidi chamber for self-insertion was inserted in a glass-bottom plate, coated with Fibronectin and cell-tracker stained MEC1 cells were seeded. Ibidi chambers allow parallel scanning in two conditions (control [1a] and CCL19-stimulated [2a] first, captured for 30 min). After that, cells were stimulated with the chemokine [1b] and one of the pools was inhibited with the CK1 inh I [2b] and scanned for additional 30 min. From the first period, MEC1 migration in control and CCL19- stimulated wells [1a, 2a] is compared to each other and after that, CCL19 only and CCL19 + CK1 inh [1b, 2b] are compared to each other. At least 50 cells was tracked in each condition. ***, P < 0.001 (Kruskal-Wallis test).

Article Snippet: After 6 h, medium was replaced by fresh one containing 20 μM CK1 inhibitor (PF-670462, Tocris), 10 μM Wnt-C59 (ab-142216, Abcam) or corresponding amount of MQ water and then the cells were incubated for another 24 h. Then they were lysed by 150 μL of 1% Laemmli buffer per well, sonicated and heated to 98°C for 2 min. VANGL2-Venus MEC1 cells were peleted (106 cells per sample, 200 × g, 5 min, RT), washed by PBS and directly lysed by 1% Laemmli lysis byffer.

Techniques: Migration, Live Cell Imaging, Staining, Time-lapse Microscopy, Clinical Proteomics, Control

Co-expression of CK1ϵ with FLAG-Dvl3 retards electrophoretic migration and induces phosphorylation-dependent shift of Dvl ( PS-Dvl3 ) ( A ) and induces TCF/LEF-dependent transcription as shown by TopFlash assay. Samples were analyzed by one-way analysis of variance followed by Tukey post tests. ***, p < 0.001, n = 3 ( B ). C , representative example of Coomassie Brilliant Blue R-stained gel used for MS/MS analysis. See the details “Experimental Procedures.”

Journal: The Journal of Biological Chemistry

Article Title: Functional Analysis of Dishevelled-3 Phosphorylation Identifies Distinct Mechanisms Driven by Casein Kinase 1ϵ and Frizzled5 *

doi: 10.1074/jbc.M114.590638

Figure Lengend Snippet: Co-expression of CK1ϵ with FLAG-Dvl3 retards electrophoretic migration and induces phosphorylation-dependent shift of Dvl ( PS-Dvl3 ) ( A ) and induces TCF/LEF-dependent transcription as shown by TopFlash assay. Samples were analyzed by one-way analysis of variance followed by Tukey post tests. ***, p < 0.001, n = 3 ( B ). C , representative example of Coomassie Brilliant Blue R-stained gel used for MS/MS analysis. See the details “Experimental Procedures.”

Article Snippet: Cells were treated by 10 μ m CK1 δ/ε inhibitor PF-670642 (Santa Cruz sc-204180).

Techniques: Expressing, Migration, Phospho-proteomics, TOPFlash assay, Staining, Tandem Mass Spectroscopy

Journal: The Journal of Biological Chemistry

Article Title: Functional Analysis of Dishevelled-3 Phosphorylation Identifies Distinct Mechanisms Driven by Casein Kinase 1ϵ and Frizzled5 *

doi: 10.1074/jbc.M114.590638

Figure Lengend Snippet: Summary of hDvl3 phosphorylations

Article Snippet: Cells were treated by 10 μ m CK1 δ/ε inhibitor PF-670642 (Santa Cruz sc-204180).

Techniques:

A , Dvl3 phosphorylation (summary of MS/MS data). Position and dynamics of detected phosphorylations is indicated by color coded lines: green , less frequent in CK1ϵ induced samples; blue , constitutive; orange , more often in CK1ϵ-induced samples; red , only in CK1ϵ-induced samples. The yellow / violet lines indicate hDvl3 protein coverage in the MS/MS analysis ( yellow , significant identification; violet , insignificant identification). C1 , C2 , C3 , C4 , Ser-280 , and Ser-311 labels indicate the position of residues mutated in the study. B , secondary structure of Dvl3 as predicted by PONDR-FIT software ( upper panel ). Structured regions are indicated by values <0.5 and unstructured by values >0.5. Accuracy of the prediction is confirmed by the identification of DIX, PDZ, and DEP domains as regions with secondary structure. In silico mutation of all identified phosphorylated Ser/Thr sites in the basic region and C terminus for Asp did not lead to the massive changes in the secondary structure ( lower panel ).

Journal: The Journal of Biological Chemistry

Article Title: Functional Analysis of Dishevelled-3 Phosphorylation Identifies Distinct Mechanisms Driven by Casein Kinase 1ϵ and Frizzled5 *

doi: 10.1074/jbc.M114.590638

Figure Lengend Snippet: A , Dvl3 phosphorylation (summary of MS/MS data). Position and dynamics of detected phosphorylations is indicated by color coded lines: green , less frequent in CK1ϵ induced samples; blue , constitutive; orange , more often in CK1ϵ-induced samples; red , only in CK1ϵ-induced samples. The yellow / violet lines indicate hDvl3 protein coverage in the MS/MS analysis ( yellow , significant identification; violet , insignificant identification). C1 , C2 , C3 , C4 , Ser-280 , and Ser-311 labels indicate the position of residues mutated in the study. B , secondary structure of Dvl3 as predicted by PONDR-FIT software ( upper panel ). Structured regions are indicated by values <0.5 and unstructured by values >0.5. Accuracy of the prediction is confirmed by the identification of DIX, PDZ, and DEP domains as regions with secondary structure. In silico mutation of all identified phosphorylated Ser/Thr sites in the basic region and C terminus for Asp did not lead to the massive changes in the secondary structure ( lower panel ).

Article Snippet: Cells were treated by 10 μ m CK1 δ/ε inhibitor PF-670642 (Santa Cruz sc-204180).

Techniques: Phospho-proteomics, Tandem Mass Spectroscopy, Software, In Silico, Mutagenesis

Phospho-preventing mutations of Ser-280/Ser-311 in the PDZ domain blocks activation of the Wnt/β-catenin pathway. A–C , HEK 293t cells were transfected with the indicated Dvl3 constructs, TopFlash reporter plasmid, plasmid encoding Renilla luciferase as an internal control, and CK1ϵ. A , mutations of Ser-280 and Ser-311 prevent efficient activation of Wnt/β-catenin by Dvl3. B , the function of Ser-280 is conserved in Dvl1 and Dvl2. Dvl1-S282A and Dvl2-S298A, corresponding to Dvl3 S280A, activated the Wnt/β-catenin pathway less efficiently than WT Dvl1 and Dvl2, respectively. C , in CK1ϵ-treated samples no significant differences between the WT Dvl3 and the mutant Dvl3 were detected. Samples were analyzed by one-way analysis of variance followed by Tukey post tests. *, p < 0.05; ***, p < 0.001; number of experiments ≥4. D , ventral overexpression of XDvl3 induced partial secondary body axes in Xenopus embryos. This phenotype reflects activation of the Wnt/β-catenin pathway in vivo . Images of one normal embryo and one embryo with a secondary axis are shown in larger magnification in addition to representative sets of embryos injected as indicated. The secondary body axes are indicated by arrows. E , the graph shows the average percentage of embryos that developed a secondary body axis from at least three independent experiments; error bars indicate the standard error , and asterisks indicate statistically significant deviation compared with overexpression of Dvl3-WT ( t test, p > 0.99). F , interaction sites of Idax (in yellow ), Fzd7 (in blue ), and Tmem88 (in green ) proteins determined for mouse Dvl1 PDZ domain (PDB ID 1MC7 ). Phylogenetically conserved Ser-280 and Ser-311 phosphorylation sites in human Dvl3 PDZ are shown in red. G , left panel , schematic representation of secondary structure of mouse Dvl1 PDZ domain. Central panel , modeled electrostatic surface potential of mDvl1 PDZ. Right panel , electrostatic surface potential of mDvl1 PDZ bearing phosphomimicking mutations at positions corresponding to Ser-280 and Ser-311 of human Dvl3 PDZ. Phosphorylation sites are highlighted in magenta ( magenta arrow ). Calculations of electrostatic surface potential were performed in UCSF Chimera software.

Journal: The Journal of Biological Chemistry

Article Title: Functional Analysis of Dishevelled-3 Phosphorylation Identifies Distinct Mechanisms Driven by Casein Kinase 1ϵ and Frizzled5 *

doi: 10.1074/jbc.M114.590638

Figure Lengend Snippet: Phospho-preventing mutations of Ser-280/Ser-311 in the PDZ domain blocks activation of the Wnt/β-catenin pathway. A–C , HEK 293t cells were transfected with the indicated Dvl3 constructs, TopFlash reporter plasmid, plasmid encoding Renilla luciferase as an internal control, and CK1ϵ. A , mutations of Ser-280 and Ser-311 prevent efficient activation of Wnt/β-catenin by Dvl3. B , the function of Ser-280 is conserved in Dvl1 and Dvl2. Dvl1-S282A and Dvl2-S298A, corresponding to Dvl3 S280A, activated the Wnt/β-catenin pathway less efficiently than WT Dvl1 and Dvl2, respectively. C , in CK1ϵ-treated samples no significant differences between the WT Dvl3 and the mutant Dvl3 were detected. Samples were analyzed by one-way analysis of variance followed by Tukey post tests. *, p < 0.05; ***, p < 0.001; number of experiments ≥4. D , ventral overexpression of XDvl3 induced partial secondary body axes in Xenopus embryos. This phenotype reflects activation of the Wnt/β-catenin pathway in vivo . Images of one normal embryo and one embryo with a secondary axis are shown in larger magnification in addition to representative sets of embryos injected as indicated. The secondary body axes are indicated by arrows. E , the graph shows the average percentage of embryos that developed a secondary body axis from at least three independent experiments; error bars indicate the standard error , and asterisks indicate statistically significant deviation compared with overexpression of Dvl3-WT ( t test, p > 0.99). F , interaction sites of Idax (in yellow ), Fzd7 (in blue ), and Tmem88 (in green ) proteins determined for mouse Dvl1 PDZ domain (PDB ID 1MC7 ). Phylogenetically conserved Ser-280 and Ser-311 phosphorylation sites in human Dvl3 PDZ are shown in red. G , left panel , schematic representation of secondary structure of mouse Dvl1 PDZ domain. Central panel , modeled electrostatic surface potential of mDvl1 PDZ. Right panel , electrostatic surface potential of mDvl1 PDZ bearing phosphomimicking mutations at positions corresponding to Ser-280 and Ser-311 of human Dvl3 PDZ. Phosphorylation sites are highlighted in magenta ( magenta arrow ). Calculations of electrostatic surface potential were performed in UCSF Chimera software.

Article Snippet: Cells were treated by 10 μ m CK1 δ/ε inhibitor PF-670642 (Santa Cruz sc-204180).

Techniques: Activation Assay, Transfection, Construct, Plasmid Preparation, Luciferase, Control, Mutagenesis, Over Expression, In Vivo, Injection, Phospho-proteomics, Software

Functional analysis of phosphorylation sites in clusters C1–C4. A , representation of Dvl structure with the positions of mutated residues. B and C , HEK293t cells were transfected with the indicated plasmids, and the electrophoretic migration of Dvl3 in the absence ( B ) and presence ( C ) of CK1ϵ was analyzed by Western blotting with anti-FLAG antibody. Mutations in C2, C2+3, and C2+3+4 blocked PS-Dvl3 induction by CK1ϵ ( C ). The protein expression levels of individual mutants were comparable. D , the ability of individual Dvl3 mutants to interact with CK1ϵ was tested by immunoprecipitation ( IP ) of CK1ϵ and FLAG-Dvl3 and subsequent analysis by Western blotting (total cell lysate (TCL)). E , HEK293t cells were transfected with the indicated plasmids, and the ability to activate TCF/LEF-dependent transcription was assessed by TopFlash system. None of the C1, C2, C2+3, C2+3+4 mutants showed any statistically significant difference. Statistical differences were analyzed by one-way analysis of variance followed by Tukey post tests. Number of experiments n ≥ 3. F and G , XDvl3 S625A/S628A/S631A (XDvl3 C2 S-A) induced secondary body axes in Xenopus embryos at the same rate as XDvl3 WT. F , the image shows a representative set of embryos injected as indicated. G , the graph summarizes the average percentage of embryos that developed a secondary body axis from at least three independent experiments; error bars indicate the standard error. n.s. , not significant. H , HEK293t cells were transfected with the indicated plasmids, and the electrophoretic mobility of Dvl3 after V5-Fzd5 coexpression was analyzed by Western blotting. With the exception of Dvl3-K435M, electrophoretic mobility of all Dvl3 mutants was indistinguishable from wild type.

Journal: The Journal of Biological Chemistry

Article Title: Functional Analysis of Dishevelled-3 Phosphorylation Identifies Distinct Mechanisms Driven by Casein Kinase 1ϵ and Frizzled5 *

doi: 10.1074/jbc.M114.590638

Figure Lengend Snippet: Functional analysis of phosphorylation sites in clusters C1–C4. A , representation of Dvl structure with the positions of mutated residues. B and C , HEK293t cells were transfected with the indicated plasmids, and the electrophoretic migration of Dvl3 in the absence ( B ) and presence ( C ) of CK1ϵ was analyzed by Western blotting with anti-FLAG antibody. Mutations in C2, C2+3, and C2+3+4 blocked PS-Dvl3 induction by CK1ϵ ( C ). The protein expression levels of individual mutants were comparable. D , the ability of individual Dvl3 mutants to interact with CK1ϵ was tested by immunoprecipitation ( IP ) of CK1ϵ and FLAG-Dvl3 and subsequent analysis by Western blotting (total cell lysate (TCL)). E , HEK293t cells were transfected with the indicated plasmids, and the ability to activate TCF/LEF-dependent transcription was assessed by TopFlash system. None of the C1, C2, C2+3, C2+3+4 mutants showed any statistically significant difference. Statistical differences were analyzed by one-way analysis of variance followed by Tukey post tests. Number of experiments n ≥ 3. F and G , XDvl3 S625A/S628A/S631A (XDvl3 C2 S-A) induced secondary body axes in Xenopus embryos at the same rate as XDvl3 WT. F , the image shows a representative set of embryos injected as indicated. G , the graph summarizes the average percentage of embryos that developed a secondary body axis from at least three independent experiments; error bars indicate the standard error. n.s. , not significant. H , HEK293t cells were transfected with the indicated plasmids, and the electrophoretic mobility of Dvl3 after V5-Fzd5 coexpression was analyzed by Western blotting. With the exception of Dvl3-K435M, electrophoretic mobility of all Dvl3 mutants was indistinguishable from wild type.

Article Snippet: Cells were treated by 10 μ m CK1 δ/ε inhibitor PF-670642 (Santa Cruz sc-204180).

Techniques: Functional Assay, Phospho-proteomics, Transfection, Migration, Western Blot, Expressing, Immunoprecipitation, Injection

Analysis of subcellular localization of individual Dvl pools. A , representation of typical distribution patterns of Dvl3. (i), punctate; (ii), even, (iii), membrane. B , HEK293t cells were transfected with corresponding plasmids and fixed, and Dvl3 was stained with anti-FLAG antibody. Distribution pattern of Dvl3 was analyzed in at least 100 cells. CK1ϵ is unable to promote even localization of Dvl3 in C2, C3, and C4 S-A mutants, whereas V5-Fzd5 changes the intracellular distribution of Dvl3 to membranous in all tested constructs with the exception of Dvl3K435M, which served as a positive control. C , HEK293t cells were transfected with corresponding plasmids and treated as indicated with 10 μ m CK1δ/ϵ inhibitor PF-670462. Inhibition of CK1δ/ϵ activity resulted in faster migration of FLAG-hDvl3 in control and CK1ϵ-induced conditions. In conditions with FLAG-hDvl3 coexpressed with V5-Fzd5 and dominant negative ( DN ) CK1ϵ-P3, acceleration of FLAG-hDvl3 electrophoretic mobility was observed only in mutant K435M. This finding confirms that CK1ϵ is and K435 is not dispensable for V5-Fzd5-induced phosphorylation of FLAG-hDvl3. D , phospho-Ser-643-Dvl3 was detected using anti-Ser(P)-643-Dvl3-specific antibody by Western blotting. Total Dvl3 was detected by anti-FLAG antibody. Anti-Ser(P)-643-Dvl3-specific antibody does not detect Dvl3 C2+C3 S-A mutant, but it strongly recognizes Dvl3 co-expressed with CK1ϵ. E , inhibition of endogenous CK1ϵ blocks signal detected by anti-Ser(P)-643-Dvl3 specific antibody. Decline in signal intensity was confirmed by Western blot quantification. CK1ϵ overexpression serves as a positive control. F , phospho-Ser-643-Dvl3 was detected by immunocytochemistry in HEK293t cells transfected with the indicated combination of plasmids. The signal of anti-Ser(P)-643-Dvl3 antibody ( red ) is negligible for Dvl3 expressed either alone or in combination with Fzd5 but strong for evenly distributed Dvl3 after CK1ϵ co-expression. Total Dvl3 detected by anti-FLAG antibody is shown in green . All confocal images were acquired using the same laser/detector settings and subsequently quantified using ImageJ software. Graphs show the overlap of fluorescence intensity peaks of individual channels along profiles indicated in the merged micrographs by a white line. A.U. , arbitrary units.

Journal: The Journal of Biological Chemistry

Article Title: Functional Analysis of Dishevelled-3 Phosphorylation Identifies Distinct Mechanisms Driven by Casein Kinase 1ϵ and Frizzled5 *

doi: 10.1074/jbc.M114.590638

Figure Lengend Snippet: Analysis of subcellular localization of individual Dvl pools. A , representation of typical distribution patterns of Dvl3. (i), punctate; (ii), even, (iii), membrane. B , HEK293t cells were transfected with corresponding plasmids and fixed, and Dvl3 was stained with anti-FLAG antibody. Distribution pattern of Dvl3 was analyzed in at least 100 cells. CK1ϵ is unable to promote even localization of Dvl3 in C2, C3, and C4 S-A mutants, whereas V5-Fzd5 changes the intracellular distribution of Dvl3 to membranous in all tested constructs with the exception of Dvl3K435M, which served as a positive control. C , HEK293t cells were transfected with corresponding plasmids and treated as indicated with 10 μ m CK1δ/ϵ inhibitor PF-670462. Inhibition of CK1δ/ϵ activity resulted in faster migration of FLAG-hDvl3 in control and CK1ϵ-induced conditions. In conditions with FLAG-hDvl3 coexpressed with V5-Fzd5 and dominant negative ( DN ) CK1ϵ-P3, acceleration of FLAG-hDvl3 electrophoretic mobility was observed only in mutant K435M. This finding confirms that CK1ϵ is and K435 is not dispensable for V5-Fzd5-induced phosphorylation of FLAG-hDvl3. D , phospho-Ser-643-Dvl3 was detected using anti-Ser(P)-643-Dvl3-specific antibody by Western blotting. Total Dvl3 was detected by anti-FLAG antibody. Anti-Ser(P)-643-Dvl3-specific antibody does not detect Dvl3 C2+C3 S-A mutant, but it strongly recognizes Dvl3 co-expressed with CK1ϵ. E , inhibition of endogenous CK1ϵ blocks signal detected by anti-Ser(P)-643-Dvl3 specific antibody. Decline in signal intensity was confirmed by Western blot quantification. CK1ϵ overexpression serves as a positive control. F , phospho-Ser-643-Dvl3 was detected by immunocytochemistry in HEK293t cells transfected with the indicated combination of plasmids. The signal of anti-Ser(P)-643-Dvl3 antibody ( red ) is negligible for Dvl3 expressed either alone or in combination with Fzd5 but strong for evenly distributed Dvl3 after CK1ϵ co-expression. Total Dvl3 detected by anti-FLAG antibody is shown in green . All confocal images were acquired using the same laser/detector settings and subsequently quantified using ImageJ software. Graphs show the overlap of fluorescence intensity peaks of individual channels along profiles indicated in the merged micrographs by a white line. A.U. , arbitrary units.

Article Snippet: Cells were treated by 10 μ m CK1 δ/ε inhibitor PF-670642 (Santa Cruz sc-204180).

Techniques: Membrane, Transfection, Staining, Construct, Positive Control, Inhibition, Activity Assay, Migration, Control, Dominant Negative Mutation, Mutagenesis, Phospho-proteomics, Western Blot, Over Expression, Immunocytochemistry, Expressing, Software, Fluorescence

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