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: Frontiers in Neuroscience

Article Title: Quantitative trait loci on chromosomes 9 and 19 modulate AII amacrine cell number in the mouse retina

doi: 10.3389/fnins.2023.1078168

Figure Lengend Snippet: Full-length and truncated Dixdc1 transcripts are differentially regulated during development. (A) The genomic structure of Dixdc1 , with conventions as in . Three transcripts are annotated in the RefSeq database, each with 20 exons and ∼77 kb in size. A/J and B6/J genomes are discriminated by many sequence variants, including one missense variant and one splice region INDEL of particular interest. The positions of two microarray probes are indicated, one probe recognizing the 3′UTR of the three annotated transcripts, and one probe that is complementary to a portion of the second intron. The positions of the qPCR primers are indicated at the very bottom. (B) Magnified region encompassing the second intron. Two transcripts that terminate within this intron were identified in the Ensembl database (release 102), one of which is recognized by the microarray probe. (C) The splice variant, a three-nucleotide INDEL, is in the second intron near the splice donor site. (D) The missense variant results in an amino acid substitution from nonpolar isoleucine to a polar threonine within the actin binding domain of Dixdc1 . B6/J carries the evolutionarily conserved sequence in each case. (E) Microarray analysis derived from adult whole eye mRNA (normalization described in ) confirms Dixdc1 expression using either the 3′UTR probe or the intronic probe in each RI strain, showing considerable (if largely uncorrelated) variation across strains. (F) Variation in the expression of truncated transcripts (ENSMUST00000118707) mapped a cis -eQTL, where the presence of the B haplotype at this locus was correlated with increased expression. (G) qPCR analysis reveals the truncated transcripts to be the dominant form in maturity, showing comparable levels of each transcript in both strains. (H) During postnatal development, the full-length transcripts are more prevalent, with expression of both transcripts increasing significantly as a function of age. Strain differences in expression were observed at later ages, yielding significant differences in the proportion of truncated to full-length transcripts, with B6/J retinas exhibiting a larger proportion than A/J retinas. (I) A luciferase expression assay compared the effectiveness of a full-length DIXDC1 isoform (NP_835219; translated from NM_001374656), an identical DIXDC1 isoform but now containing the missense mutation, and a truncated DIXDC1 isoform similar to that expected to be translated from the truncated transcripts, upon DVL2-mediated β-catenin signaling. Functional domains were determined from UniProt ( uniport.org ) annotations; CH, calponin homology domain; AB, actin binding domain; CC, coiled coil domain; DIX, DIX domain. (J) The missense isoform showed no functional effect of the mutation, augmenting β-catenin signaling to similar levels as full-length DIXDC1, while the truncated isoform was ineffective at modulating β-catenin signaling in either direction, with luciferase expression levels identical to controls. n = the number of adult retinas in panel (F) , the number of pooled samples from postnatal retinas in panel (G) , and the number of wells in panel (H) .

Article Snippet: In addition to the DIXDC1 expression plasmids, a plasmid that expresses Disheveled 2 (DVL2) under a CMV promoter (#123587; Addgene; RRID:Addgene_123587 ) was also used to stimulate WNT signaling in transfected cells ( ).

Techniques: Sequencing, Variant Assay, Microarray, Binding Assay, Derivative Assay, Expressing, Luciferase, Mutagenesis, Functional Assay