Fig. 3: Q84Pfs fragment (Q86Pfs for human) is expressed in Q84Pfs-Het mouse cortex and interacts with FOXG1-fl. | Nature Communications

Fig. 3: Q84Pfs fragment (Q86Pfs for human) is expressed in Q84Pfs-Het mouse cortex and interacts with FOXG1-fl.

From: The patient-specific mouse model with Foxg1 frameshift mutation provides insights into the pathophysiology of FOXG1 syndrome

Fig. 3

a, b The immunostaining analyses of E16 Q84Pfs-Het and WT cortices with FOXG1-N-Ab and FOXG1-C-Ab. The fluorescence intensity in individual cells was quantified in (b). The signal intensity with FOXG1-N-Ab was increased in Q84Pfs-Het relative to WT (**p = 0.0011), but the signal intensity with FOXG1-C-Ab was significantly lower in Q84Pfs-Het than WT (****p < 0.0001). The mean intensity was measured in three independent sections per mice (n = 3 mice/condition). The midline represents the mean. Two-tailed Mann-Whitney test was used. Scale bars, 25 μm (a set of images on the left) or 10 μm (a set of magnified images on the right). c CoIP assays in HEK293T cells transfected with Flag-tagged FOXG1-fl and HA-tagged FOXG1-fl (the left set) or with FOXG1-fl and Flag-tagged Q86Pfs (the right set). In the coIP set on the left, the association between Flag-FOXG1-fl and HA-FOXG1-fl was tested by immunoprecipitation with Flag antibody, followed by western blot with HA antibody. In the coIP set on the right, the association between FOXG1-fl and Flag-Q84Pfs was monitored by immunoprecipitation with Flag antibody, followed by western blot with FOXG1-N-Ab that detects both FOXG1-fl and Q84Pfs and distinguishes the two proteins by the protein size differences. CoIP data show that FOXG1-fl interacts with each other and with Q86Pfs fragment. d The immunostaining analyses in HEK293T cells transfected with FOXG1-fl alone (top panel), Q86Pfs alone (middle panel), and both FOXG1-fl and Q86Pfs (bottom panel). GFP labels the transfected cells as all plasmids have ires-GFP sequences. The subcellular distribution of FOXG1-fl and Q86Pfs proteins was assessed using FOXG1-N-Ab and FOXG1-C-Ab. FOXG1-fl protein, detected by both antibodies, is localized in nuclei, whereas Q86Pfs, recognized by only FOXG1-N-Ab but not by FOXG1-C-Ab, shows prominent speckles in nuclei and cytosol. When co-expressed, FOXG1-fl is localized in Q86Pfs+ nuclear speckles (white arrows). Scale bars, 5 μm. e The model. In Q84Pfs-Het neurons, Q84Pfs proteins are co-expressed and form speckle-like patterns with FOXG1-fl. In WT neurons, FOXG1 is primarily expressed in nuclei. Only the representative images are shown. The experiments repeated three (c) and six times (d) independently.

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