Fig. 1: Mutant TTC7A is depleted from the nucleus and fails to bind with chromatin. | Cell Discovery

Fig. 1: Mutant TTC7A is depleted from the nucleus and fails to bind with chromatin.

From: Tetratricopeptide repeat domain 7A is a nuclear factor that modulates transcription and chromatin structure

Fig. 1

a Subcellular fractionation of B lymphoblastoid cell lines (B-LCLs) from three controls and patients (P1-2-3_E71K). TTC7A (96 kDa) expression level was assessed in the cytoplasmic and nuclear fractions by western blot. α-tubulin and the total histone H2B were used to monitor the purity of the isolated fraction. b Immunoprecipitation of TTC7A protein in the cytoplasm (Cyto), and nuclear (Nucl) fraction using TTC7A or control IgG antibodies in three controls (Ctr) and three patients (P 1 to 3 _E71K) in B-LCLs. Immunoprecipitated fractions were resolved by western blot and sequenced by mass spectrometry. Abundance of TTC7A was scored after normalization. The mean score  ± SEM are indicated. c Cells expressing WT_ or E71K_TTC7A-HA were imaged on the ImageStream system and analyzed for HA-Alexa Fluor 488 and DAPI staining with IDEAS software. Correlation between WT (blue) and E71K_TTC7A (red) signals was measured using a similarity score. A mean similarity score for histone H3 staining and DAPI is provided as a positive control. Mean of three independent experiments, total number of cells in WT = 1723 and E71K = 2574, and unpaired t test p-value < 0.0001. d TTC7A cellular localization in B-LCLs by confocal microscopy. Cells expressing WT_ or E71K_TTC7A-HA were stained with HA (green) and DAPI (blue). Scale bar: 5 µm. e Isolated subcellular fractionation were subjected to western blot and assessed for TTC7A level and specific antibodies for each compartment. Cytoplasm (Cyt), membrane (Mb), nuclear matrix (N.M), and chromatin-bound (chr). f Control cells were transduced with WT or E71K TTC7A-HA-ires-GFP. Stably expressing cells were subjected to subcellular fractionation, similar to (c)

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