Journal: NAR Molecular Medicine

Article Title: In silico and in vitro mutational analysis to investigate the structure and pathogenicity of POT1 missense variants

doi: 10.1093/narmme/ugaf004

Figure Lengend Snippet: Telomeres, the shelterin complex, POT1, and missense POT1 mutations of interest. ( A ) Telomeres and the shelterin complex protein. ( B ) Canonical hPOT1 domains, the POT1N and POT1C PDB structures included in this study, along with the 14 selected missense POT1 mutations in MD. ( C ) Case-reported missense POT1 variant distribution tabulated from the ClinVar database, NCCS CGS patients, and ( D ) SG10K_Health population, in comparison to the current ACMG classifications for missense POT1 variants. Nonclassified reported variants (in gray) are representative.

Article Snippet: The primary antibodies in use are c-Myc (mouse, Thermo Fisher #13-2500), POT1 (mouse, Merck #MABE1907), and FLAG (mouse, Sigma #1804).

Techniques: Variant Assay, Comparison

Journal: NAR Molecular Medicine

Article Title: In silico and in vitro mutational analysis to investigate the structure and pathogenicity of POT1 missense variants

doi: 10.1093/narmme/ugaf004

Figure Lengend Snippet: Missense POT1 variant prioritization.

Article Snippet: The primary antibodies in use are c-Myc (mouse, Thermo Fisher #13-2500), POT1 (mouse, Merck #MABE1907), and FLAG (mouse, Sigma #1804).

Techniques: Variant Assay

Journal: NAR Molecular Medicine

Article Title: In silico and in vitro mutational analysis to investigate the structure and pathogenicity of POT1 missense variants

doi: 10.1093/narmme/ugaf004

Figure Lengend Snippet: Multiple sequence alignment among POT1 protein homologs. Color gradient was based on percentage identity using Jalview.

Article Snippet: The primary antibodies in use are c-Myc (mouse, Thermo Fisher #13-2500), POT1 (mouse, Merck #MABE1907), and FLAG (mouse, Sigma #1804).

Techniques: Sequencing

Journal: NAR Molecular Medicine

Article Title: In silico and in vitro mutational analysis to investigate the structure and pathogenicity of POT1 missense variants

doi: 10.1093/narmme/ugaf004

Figure Lengend Snippet: ( A ) EMSA to analyze the ability of in vitro -translated POT1 proteins bind to telomere oligonucleotide. ( B ) Western blotting results of in vitro translated products show POT1 protein was translated from plasmid at similar efficiency, with the Myc antibody confirming specificity. ( C ) Quantification for POT1–telomere oligo binding EMSA readouts. Four independent experiments were conducted, and Student’s t -test was employed to demonstrate statistical difference among the readout. ( D ) TIF analysis to detect the presence of DNA repair proteins at sites of telomere dysfunction based on the co-localization of DNA damage markers 53BP1 and shelterin protein TRF1. HEK-293T-POT1-KO cells stably expressed POT1 mutants WT, EV, I115T, D224N, H266Q, S270N, P371T, T416A, P601L, and Q623H were examined for colocalization of 53BP1 (red) and TRF1 (green) to identify TIF signals. Representative IF images show “TIF-negative” nuclei in WT and “TIF-positive” nuclei in EV. ( E ) Quantification is shown for the ratio of cells with at TIF-positive signals to total cell count. At least 150 nuclei were counted for each sample. ( F ) Coimmunoprecipitation (Co-IP) assay to assess the binding of POT1 mutant to TPP1. HEK-293T-POT1-KO WT, EV, and mutant cell lines (P371T, G404V, T416A, P601L, and Q623H) were transfected with 5 μg of TPP1 (also known as ACD). Cell lysates were incubated with Flag antibody and Dynabead Protein G followed by western blot analysis. The blot shows the levels of POT1 binding to FLAG-TPP1 in the indicated stable cell lines.

Article Snippet: The primary antibodies in use are c-Myc (mouse, Thermo Fisher #13-2500), POT1 (mouse, Merck #MABE1907), and FLAG (mouse, Sigma #1804).

Techniques: In Vitro, Western Blot, Plasmid Preparation, Binding Assay, Stable Transfection, Cell Counting, Co-Immunoprecipitation Assay, Mutagenesis, Transfection, Incubation