Figure 1: Distribution of Gβ2 protein on the surface of mitochondria.
From: G-protein β2 subunit interacts with mitofusin 1 to regulate mitochondrial fusion
(a) Immunostaining with a specific antibody shows the localization of Gβ2 in HeLa cells. Cells were stained with 20 nM Mitotracker Red, and then immuno-labelled with anti-Gβ2 (green) and visualized with a two-photon microscope (Zeiss, LSM 510). The boxed area on the upper left was scanned along the Z axis, and the images were superposed to form the three-dimensional (3D) image. The boxed area in the 3D image is shown at higher magnification in the upper right panels. The lower right shows images of Y to Z dimension. Scale bar, 20 μm. (b) Distribution of Gβ1, Gα12, Gαi1 and Gγ2 in HeLa cells. Representative images of HeLa cells labelled with anti-Gβ1, Gα12, Gαi1 and Gγ2 antibodies (green) and stained with 20 nM Mitotracker Red. Scale bar, 10 μm. (c) Distribution of Gβ2 and other G proteins in the mitochondrial fraction. Equal amounts of the whole cell lysate (W), cytosol (C1), light membrane (C2), mitochondria (M) and plasma membrane (P) fractions were loaded and analysed for the distribution of Gβ2, Gγ2 and other G proteins by western blot using specific antibodies. The purity of isolated mitochondria was determined by using the markers for distinct organelles: Mfn1, VDAC1 (mitochondria), GM130 (Golgi), p62 (endoplasmic reticulum) and actin (cytoplasm). (d) Protease K assay for Gβ2's localization on mitochondria. Mitochondrial fractionation obtained from HeLa cells was subjected to Protease K digestion for 30 min with different concentrations as indicated. Gβ2 and other mitochondrial proteins such as Mfn1, Tom20 (outer mitochondrial membrane, OMM) and Tim23 (inner mitochondrial membrane, IMM) were analysed by western blot with corresponding antibodies. Equal amounts of protein were loaded for Protease K treatment. (e) Gβ2 is the most abundant subunit of Gβ proteins in HeLa cells. mRNA levels of distinct Gβ subunits were analysed by semiquantitative PCR in HeLa cells. (f, g) The mRNA levels of Gα, Gγ subunits in the cells were analysed by semiquantitative PCR (right panel). The size markers are 1,000, 750, 500, 200 and 100 bp, respectively, from top to bottom. All the experiments were repeated at least three times.
