immunoblotting  (Proteintech)

Journal: Nature Communications

Article Title: Proximity analysis of native proteomes reveals phenotypic modifiers in a mouse model of autism and related neurodevelopmental conditions

doi: 10.1038/s41467-024-51037-x

Figure Lengend Snippet: A Schematic illustration of HiUGE-iBioID and its workflow. GS-sgRNA: gene-specific-gRNA. DS-sgRNA: donor-specific-gRNA. Strategies to preserve C-term PDZ-binding motifs of Syngap1, Ctnnb1, Iqsec2, and Lrrc4c are detailed. B Overview of 14 proximity proteomes that segregates according to expected bait functions. C Enrichment analysis of overlapping SFARI genes using hypergeometric probability. The solid red line denotes Bonferroni adjusted p-value at 0.05. D Proximity proteome clustering based on a similarity matrix. E Core proximity proteome between Syngap1 and Anks1b that show highly significant overlaps with SFARI genes. F Core proximity proteome amongst Ank3, Scn2a, and Scn8a that are clustered based on similarity. Modules of proteins were isolated by MCL clustering or GO analysis.

Article Snippet: Expression vectors of Myc-DDK-tagged human ORF clones of ANKS1B and SYNGAP1 were purchased from Origene (#RC211877, #RC229432).

Techniques: Binding Assay, Isolation

Journal: Nature Communications

Article Title: Proximity analysis of native proteomes reveals phenotypic modifiers in a mouse model of autism and related neurodevelopmental conditions

doi: 10.1038/s41467-024-51037-x

Figure Lengend Snippet: A Schematic illustration of the quantitative proteomic characterization of Syngap1-Het synaptosomes. B Proteomic alterations identified in the Syngap1-Het synaptosome. Proteins that overlap with the Syngap1 proximity proteome are underlined. C Co-immunoprecipitation result showing loss of interaction with ANKS1B in frame-shifting c.2214_2217del SYNGAP1 mutation. D HiUGE labeling of truncated Syngap1 at exon 13 shows synaptic localization (boxed region) and aberrant somatic mis-localization (arrowhead). Scale bar in the enlarged view represents 2 μm. E Schematic illustration of labeling truncated Syngap1 with TurboID by targeting exon 13. F Western blot showing TurboID-HA labeled Syngap1 truncation at the expected molecular mass. G Proximity proteomic network showing conserved and neomorphic interactions in Syngap1 truncation. Note that interaction with Anks1b is no longer detected. H Schematics assessing phenotypes of the Syngap1-Anks1b functional interaction. I Western blot confirming disruption of Syngap1 and Anks1b expression. C , D , I Representative experiments are based on three replicates with similar results. J Representative raster plots of neural activities at DIV-08, 11, and 14. K – M Neurometrics showing further depletion of Anks1b exacerbates the development of precocious neural activity associated with Syngap1-LOF. * p < 0.05; ** p < 0.01; *** p < 0.001; n.s.: non-significant. One-way ANOVA followed by post-hoc Tukey HSD tests ( n = 36 wells). Plots are mean ± SEM.

Article Snippet: Expression vectors of Myc-DDK-tagged human ORF clones of ANKS1B and SYNGAP1 were purchased from Origene (#RC211877, #RC229432).

Techniques: Immunoprecipitation, Mutagenesis, Labeling, Western Blot, Functional Assay, Disruption, Expressing, Activity Assay