3xflag wt dvl2  (Addgene inc)

Journal: PLOS ONE

Article Title: Regulation of YAP and Wnt signaling by the endosomal protein MAMDC4

doi: 10.1371/journal.pone.0296003

Figure Lengend Snippet: (A) HEK293 cells over-expressing MAMDC4 were stimulated with control or Wnt3a conditioned media. Lysates were immunoprecipitated with antibodies against MAMDC4 or control IgG. MAMDC4 associates with LRP6, p-LRP6, total β-catenin, DVL2 and Axin in control and Wnt3a stimulated conditions. There is a substantial amount of LRP6 and p-LRP6 (relative to input) associated with MAMDC4 in both Wnt stimulated and unstimulated conditions. (B) Quantification of signal from immunoprecipitation relative to input. LRP6 and p-LRP6 have a ratio of approximately 1 while the ratio of β-catenin and Axin1 have a ratio of IP to input that is <1/10 th of the amount of LRP6 and p-LRP6 immunoprecipitated. All immunoprecipitations were repeated 3 to 5 times and the input lanes represents 1/50 th of lysate used for the immunoprecipitation.

Article Snippet: Cells were transfected with 3XFLAG (WT) DVL2 (cat 24803; addgene; [ ]) using Lipofectamine 2000 (cat. 1168500; ThermoFisher).

Techniques: Expressing, Control, Immunoprecipitation

Journal: PLOS ONE

Article Title: Regulation of YAP and Wnt signaling by the endosomal protein MAMDC4

doi: 10.1371/journal.pone.0296003

Figure Lengend Snippet: (A) SKCO cells were transfected with MAMDC4 and DVL2. 48 hours post transfection cultures were treated with vehicle or Wnt3a for 1hr. Cells were labeled with antibodies to MAMDC4 (green) and DVL2 (red) and counterstained with DAPI. MAMDC4 and DVL2 are colocalized in puncta regardless of Wnt3a stimulation Scale bar : 10μm. (B) Quantification of Pearson’s Correlation Coefficient (r). Wnt3a treatment does not influence MAMDC4 and DVL2 colocalization. (C) SKCO cells were transfected with MAMDC4 and Axin1. 48 hours post transfection cultures were treated with vehicle or Wnt3a for 5min. Cells were labeled with antibodies to MAMDC4 (green) and Axin1 (red) and counterstained with DAPI. MAMDC4 and Axin1 are colocalized in some puncta after Wnt3a stimulation (arrows). (D) Quantification of Pearson’s Correlation Coefficient (r). There is an increase in MAMDC4 and Axin1 colocalization that is dependent on Wnt3a stimulation. (E) Immunoblot of NeutrAvidin Pull down. (F) Quantification of LRP6 Pull down / Lysate ratio.

Article Snippet: Cells were transfected with 3XFLAG (WT) DVL2 (cat 24803; addgene; [ ]) using Lipofectamine 2000 (cat. 1168500; ThermoFisher).

Techniques: Transfection, Labeling, Western Blot

Journal: PLOS ONE

Article Title: Regulation of YAP and Wnt signaling by the endosomal protein MAMDC4

doi: 10.1371/journal.pone.0296003

Figure Lengend Snippet: SKCO cells were co-transfected with either MAMDC4 or MAMDC4 mutants and DVL2. 48 hours post transfection cells were labeled with antibodies to MAMDC4 (green) and DVL2 (red) and counterstained with DAPI. (A) MAMDC4 and DVL2 are colocalized in puncta, however, DVL2 has diffuse labeling in cells lacking MAMDC4 overexpression (arrow) (B) Quantification of number of cells expressing both MAMDC4 and DVL2, which contain DVL2 puncta. 35 to 45 cells were assessed for each condition, n = 3. **P<0.001, ***P<0.001. Statistical significance was determined using an Ordinary one-way ANOVA. Scale bar : 10μm.

Article Snippet: Cells were transfected with 3XFLAG (WT) DVL2 (cat 24803; addgene; [ ]) using Lipofectamine 2000 (cat. 1168500; ThermoFisher).

Techniques: Transfection, Labeling, Over Expression, Expressing

Journal: BMC Biology

Article Title: DIX domain containing 1 (DIXDC1) modulates VEGFR2 level in vasculatures to regulate embryonic and postnatal retina angiogenesis

doi: 10.1186/s12915-022-01240-3

Figure Lengend Snippet: DIXDC1 upregulates VEGFR2 via Dvl2. A DIXDC1 or control vector, and VEGFR2 was co-transfected in HEK293T. Immunoprecipitation with antibody against DIXDC1 and VEGFR2 result revealed that no direct interaction between DIXDC1 and VEGFR2. B DIXDC1 or control vector, and Dvl2 was co-transfected in HEK293T. Immunoprecipitation with antibody against DIXDC1 and Dvl2 result revealed that there is an interaction between DIXDC1 and Dvl2. C Endogenous immunoprecipitation with antibodies against Dvl2 and VEGFR2 and there is an interaction between the two proteins in HUVEC. D Control siRNA or siDIX1 is transfected to HUVEC and subjected to immunoprecipitation antibody against Dvl2. E, F, G, and H Quantification of relative intensity of immunoprecipitated VEGFR2, Dvl2, and input VEGFR2 and Dvl2 of D . I DIXDC1 was overexpressed and there was a positive correlation between the expression level of DIXDC1 and Dvl2. J Quantification of relative intensity of Dvl2 in I . K DIXDC1, Dvl2, and VEGFR2 were co-overexpressed in HEK293T and the expression level of VEGFR2 and Dvl2 was observed. L Quantification of relative intensity of VEGFR2 in K . All Experiments were repeated at least 4 different sets. *p < 0.05, **p < 0.005, and ***p < 0.0001, by paired, 2-tailed Student’s t test and one-way ANOVA. Error bars represent the mean ± SD. Individual values can be found in Additional file : Fig 5

Article Snippet: The Dvl2 ORF-containing plasmid 3XFlag-Dvl2(Item #24802) was purchased from Addgene (Cambridge, MA, USA). pEGFP-C2 was used as a transfection control (Clontech).

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

Journal: BMC Biology

Article Title: DIX domain containing 1 (DIXDC1) modulates VEGFR2 level in vasculatures to regulate embryonic and postnatal retina angiogenesis

doi: 10.1186/s12915-022-01240-3

Figure Lengend Snippet: DIXDC1 increases wnt/ β-catenin signaling and VEGFR2 stability via Dvl2. A knockdown of DIXDC1 affects the VEGF-induced signaling pathways (40 ng/ml) in HUVEC. B, C, and D Quantification of relative intensity of p-VEGFR2(Y1175), P-ERK1/2, and p-FAK of A . E Knockdown of DIXDC1 affects the wnt/β-catenin signaling pathways (200 ng/ml wnt3a) in HUVEC. F, G, H, and I Quantification of relative intensity of VEGFR2, non-p-β-catenin, β-catenin, and Dvl2 of E . J Interaction between Dvl2 and VEGFR2 is affected by DIXDC1 and wnt3a (200 ng/ml) in HUVEC. K, L Quantification of relative intensity of immunoprecipitated VEGFR2 and Dvl2. M Suppression of DIXDC1 affects the level of HIF1a in oxygen glucose-deprived condition in HUVEC. N, O, P, Q, R, and S Quantification of relative intensity of VEGFR2, HIF1a, non-p-β-catenin, β-catenin, Dvl2, and DIXDC1 of M . T VEGFR2, Dvl2, and DIXDC1 transfected to HEK293T and treated with MG132. Accumulation of VEGFR2 level was observed with MG132. U Quantification of relative intensity of VEGFR2 of T . All Experiments were repeated at least 4 different sets. *p < 0.05, **p < 0.005, and ***p < 0.0001, by one-way ANOVA. Error bars represent the mean ± SD. Individual values can be found in Additional file : Fig. 6

Article Snippet: The Dvl2 ORF-containing plasmid 3XFlag-Dvl2(Item #24802) was purchased from Addgene (Cambridge, MA, USA). pEGFP-C2 was used as a transfection control (Clontech).

Techniques: Knockdown, Protein-Protein interactions, Immunoprecipitation, Transfection