Journal: Nucleic acids research

Article Title: The BAF A12T mutation disrupts lamin A/C interaction, impairing robust repair of nuclear envelope ruptures in Nestor-Guillermo progeria syndrome cells.

doi: 10.1093/nar/gkac726

Figure Lengend Snippet: Figure 3. The BAF A12T mutation causes BAF mislocalization in NGPS patient cells without affecting its phosphorylation by VRK1 in vitro. (A) Representative immunofluorescence images showing endogenous BAF staining in Control, NGPS patient cells and NGPS2 WT clone1; scale bar: 20 m. (B) Quantification of the mean BAF nuclear to cytoplasmic ratio in 425, 495, 401 and 363 cells for Control, NGPS1, NGPS2 and NGPS2 WTclone1, respectively. Data were obtained from three independent experiments and quantified using a one-way ANOVA analysis with ˇS´ıd´ak’s multiple comparisons test (****P<0.0001). (C) Representative immunoblot showing the expression level of BAF in whole cell lysates from the indicated cell lines. Tubulin was used as a loading control. (D–E) 2D NMR 1H-15N Heteronuclear Single Quantum Coherence (HSQC) spectra were recorded on purified BAF WT (in shades of blue) and A12T (in shades of red) upon phosphorylation by the VRK1 kinase in vitro. Six spectra are superimposed, corresponding to: non- phosphorylated BAF WT and A12T (t = 0 min), in dark and light grey respectively, mono-phosphorylated BAF WT and A12T (t = 15 min), in light blue and pink respectively, and di-phosphorylated BAF WT and A12T (t = 8 h), in dark blue and red, respectively. The zoom in (E) shows the spectral regions of the 1H-15N HSQC of BAF WT and A12T spectra where signals of phosphorylated serines and threonines are commonly observed. The signals of the two BAF residues phosphorylated by VRK1 (Ser4 and Thr3) are annotated, and the arrow indicates the shift of the peak corresponding to phosphorylated Ser4 upon phosphorylation of Thr3.

Article Snippet: The Vaccinia Related Kinase 1 (VRK1) expression vector is a gift from John Chodera, Nicholas Levinson and Markus Seeliger (Addgene plasmid #79684 (46)).

Techniques: Mutagenesis, Phospho-proteomics, In Vitro, Immunofluorescence, Staining, Control, Western Blot, Expressing, Purification