Fig. 3: Effects of EMLs on localization of IP3R. | Communications Biology

Fig. 3: Effects of EMLs on localization of IP3R.

From: ER-mitochondria distance is a critical parameter for efficient mitochondrial Ca2+ uptake and oxidative metabolism

Fig. 3

a–d Proximity ligation assay (PLA) using anti-pan-IP3R, and anti-VDAC1/3 antibody (green) of HeLa cells expressing control plasmid (ER-mRFP) and ER-mitochondrial linkers (EMLs) (red). a Cartoon illustrating that only IP3Rs and VDAC1/3 juxtaposed at ≤40 nm are labeled. Representative images (b), quantification of the PLA-labeled area intensity (c) and PLA-EML Pearson colocalization coefficient (d). Scale bars = 20 µM. e–g Immunofluorescence analysis using anti-pan-IP3R antibody (green) in ER-mRFP and EML-expressing HeLa cells (red). e Cartoon illustrating that all IP3Rs present in the cells are labeled. Representative images (f) and quantification of IP3R-EML Pearson colocalization coefficient (g). Scale bars = 20 µM. h–j Localization of endogenous IP3R1 tagged with EGFP in HeLa cells (endogenous IP3R1-EGFP, green) upon expression of ER-EBFP, and 10nm- and 20nm-EMLs containing EBFP (magenta). h Cartoon illustrating that all endogenous IP3R1 receptors are tagged with EGFP. Representative images (i) and quantification of EGFP-EML Pearson colocalization coefficient (j). Scale bars = 20 µM. Whisker plots report data collected from 54–106 (c), 25–30 (d), 8–18 (g), 16–18 (j) cells from at least 3 independent coverslips analyzed from 3 independent cultures. One-way ANOVA, Tukey post hoc test. **p < 0.01, ***p < 0.001, ****p < 0.0001.

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