PMC:3216504 / 42761-45173
Annnotations
{"target":"https://pubannotation.org/docs/sourcedb/PMC/sourceid/3216504","sourcedb":"PMC","sourceid":"3216504","source_url":"https://www.ncbi.nlm.nih.gov/pmc/3216504","text":"(A) Structure of the ZIC2 protein. Gray boxes with numbers indicate C2H2 motifs in the zinc finger domain of ZIC2. The positions of the A95T, R409P, and S444R mutations are indicated. Multiple alignments of the flanking regions of three mutations are indicated along with the reference sequences. Shaded characters show conserved cysteine and histidine residues in the C2H2 zinc fingers. Black box indicates an evolutionary conserved domain (ZOC, Zic-Opa-Conserved) domain. Gray characters indicate the mutated residues and the corresponding residues in other species (Hs, Homo sapiens [human]; Mm, Mus musculus [mouse]; Dr, Danio rerio [zebrafish]; Lb, Loligo breekeri [squid]; Dm, Drosophila melanogaster (fly); Nv, Nematostella vectensis [sea anemone]). (B) Immunoblotting of mouse wild-type Zic2 and Zic2 variants. NIH3T3 cells were transfected with the FLAG-tag expression plasmids. The Zic2 proteins were detected by the anti-FLAG antibody. Arrow indicates the fast migrating component in FLAG-Zic2-S444R. (C) NIH3T3 cells were transfected with a Zic2-responsive luciferase reporter vector together with a vector expressing wild-type FLAG-Zic2 (WT), or the FLAG-Zic2-A95T, -R409P, -S444R mutant proteins. All luciferase activities were normalized to the activities of the co-transfected elongation factor 1 promoter-driven Renilla luciferase. The means ± SEM of three independent experiments of three samples each are shown. (D) Gel mobility shift assay. IRD-labeled target DNAs were incubated with partially purified FLAG-Zic2-WT or FLAG-Zic2-R409P proteins expressed in 293T cells. The probes and the amount are indicated at the top. (E) Quantification of the gel shift assay result. Data are presented as means ± SEM. There were statistically significant differences between the FLAG-Zic2-WT and FLAG-Zic2-R409P-bound DNA probes at each dose (50 fmol, P \u003c 0.001; 100 fmol, P = 0.0022; 200 fmol, P = 0.012; and 400 fmol, P = 0.0089). (F) FLAG-Zic2-WT or FLAG-Zic2-R409P expressed in 293T cells were immunoprecipitated with the anti-FLAG antibody. Proteins in the input cell lysates (input) and immunoprecipitates (IP) were analyzed by immunoblotting using anti-DNA-PK, anti-RHA, and anti-FLAG antibodies. There was a decrease in the amount of co-precipitated DNA-PK in FLAG-Zic2-R409P immunoprecipitates compared to those from FLAG-Zic2-WT, despite comparable amounts of FLAG-Zic2-R409P and FLAG-Zic2-WT.","tracks":[]}