Fig. 2: Demonstration of ARL5-ARMH3 interactions using mitochondria-relocalization and yeast-two hybrid assays.

a Mitochondria-relocalization assays. WT HeLa cells were transiently co-transfected with constructs encoding GFP (control) or GFP-ARMH3 (green) and the indicated Mito-ARL5 constructs (magenta). Cells were then fixed, permeabilized, immunolabeled for BioID2, and imaged by confocal fluorescence microscopy. Single-channel images are shown in grayscale with nuclei (DAPI) in blue. Scale bars: 10 μm. Notice the relocalization of GFP-ARMH3 to mitochondria by the Mito-ARL5-Q70L but not -T30N constructs. b Pearson’s correlation coefficient (PCC) for the co-localization of GFP-ARMH3 or GFP with the indicated Mito-ARL5 constructs calculated from an experiment such as that shown in panel a. The graph represents the mean ± SD of values from the following number of cells in the experiment (from left to right): n = 5, 4, 5, 4, 10 and 4. The statistical significance of the differences was calculated by one-way ANOVA with multiple comparisons using Tukey’s test. P-values are indicated on the graphs. c Y2H analysis of the interaction of the constructs indicated on top fused to the GAL4 DNA-binding domain (BD) and the constructs indicated on the left fused to the GAL4 transcriptional activation domain (AD). The p53 and T-Ag fusion proteins were used as controls. Growth in the absence of histidine (−His) and presence of 3 mM 3-amino-1,2,4 triazole (3AT) is indicative of interactions. Growth in the presence of histidine (+His) is a control. Notice the interaction of ARMH3 with the WT and Q70L forms of ARL5A and ARL5B, and the failure of ARMH3 to interact with the active forms of ARL1 and ARFRP1. d Quantification of the colony area from the Y2H assays shown in panel c. The graph represents the mean ± SD from three independent, color-coded experiments. The statistical significance of the differences was calculated by one-way ANOVA with multiple comparisons to the interaction between p53 and ARMH3 (control) using Dunnett’s test. P-values are indicated on the graphs. Source data are provided in the Source Data file.