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Fig. 5 | BMC Biology

Fig. 5

From: The interactome of CLUH reveals its association to SPAG5 and its co-translational proximity to mitochondrial proteins

Fig. 5

CLUH proximity to mitochondrial proteins occurs in the cytosol and requires the TPR domain. A Western blot showing the expression of CLUH in HCT116 cells wild type (WT) and knockout for CLUH (CLUH KO). Indicated proteins are revealed using specific antibodies. B, C Western blot analysis of BioID experiments performed on CLUH KO cells (B) and on WT HCT116 cells (C) transduced to stably express BioID2-3xHA-CLUH, BioID-3xHA-CLUH∆TPR, or BioID2-3xHA-GFP proteins. The proximity labeling is performed for 24 h in the presence of 50 μM biotin in the culture medium. Biotinylated proteins are pulled down (PD) from total input extracts (INP) using streptavidin-coupled magnetic beads. The loaded samples correspond to 0.5% of the input and 20% of the pulled-down samples. Replicate experiments are identified with #1 and #2. The constructs are revealed with anti-HA antibody (HA). CPMPs (LRPPRC, IMMT, HSPA9 and ATP5A) and other indicated proteins are revealed using specific antibodies. The size of the endogenous CLUH (black), wildtype transgene (orange) and the delta-TPR mutant (red) is indicated. Biotinylated proteins are revealed using HRP-coupled streptavidin. D, E Immunofluorescence confocal imaging on HCT116 fixed cells showing the subcellular localization of CLUH (D) and three CLUH proximal proteins (E). The endogenous proteins are detected using specific primary antibodies and revealed using Alexa488-coupled secondary antibodies (green). Mitochondria (red) are labeled using MitoTracker™ Red CMXRos. Nuclei (blue) are stained with Hoechst. CLUH KO cells are used as control. The fluorescence profile of the red and green channel over the indicated pixel lines are shown on the right. The fluorescence signal is normalized to the highest value for each channel. F Schematic representation of the experimental workflow to obtain both “crude” and “pure” mitochondria. G Western blot on total “crude” and “pure” mitochondrial fractions from wild-type and CLUH KO HCT116 cells. Indicated proteins are revealed using specific antibodies. H Scatter plot showing the abundance (specific spectral counts) of nuclear and mitochondrial encoded proteins identified by MS from pure mitochondrial fraction (red), BioID2-CLUH (orange) and GFP-BioID2 (gray) samples. Each dot represents a biological replicate sample. The full datasets are available in Table S6

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