Anti tom20 antibody

The cells were seeded on coverslips at a density of 40,000 cells/well and transfected with FAM-labeled mmu-CR805 as described above. Twenty-four hours after transfection, the cells were fixed with 3.8% of paraformaldehyde for 20 min at room temperature, washed three times with PBS, and permeabilized with 0.05% of saponin-PBS. Non-specific staining was blocked by incubation with 5% of goat serum in 0.05% of saponin-PBS for 30 min at room temperature before incubating the cells for 2 h at room temperature with anti-Tom20 antibodies (Cell Signaling Technology, cat. #CS42406, at dilution 1:300). The cells were again washed three times, incubated with secondary antibodies conjugated to Alexa Fluor 568 (Invitrogen, cat. #A-11004, at dilution 1:500) for 1 h at room temperature, and washed twice with 0.05% of saponin-PBS. Nuclei were stained with Hoechst 33258 (Sigma-Aldrich, cat. #14530), and the cells were washed once with 0.05% of saponin-PBS and once with PBS and mounted on slides using Gold Antifade mounting medium (Thermo Fisher, cat. #P36930).

The samples of mitochondrial lysates (1 mg/mL) dissolved in Laemmli buffer (Bio-Rad, Hercules, CA, USA) were added to each line at 20 μg and divided by electrophoresis (12.5% SDS-PAGE). The proteins were transferred from the gel onto a nitrocellulose membrane (0.2 µm). The membranes were blocked in Roti-block solution (Carl Roth GmbH + Co., Karlsruhe, Germany) for 1 h. The membranes were stained with the polyclonal antibodies to DRP1 (Elabscience, Houston, TX, USA) at dilution 1:2000, Mfn2 (Elabscience, Houston, TX, USA) at dilution 1:2000, OPA1 (Cloud-Clone Corp., Katy, TX, USA) at dilution 1:1000, PHB2 (Cusabio, Houston, TX, USA) at dilution 1:1000, and PINK1 (Cusabio, Houston, TX, USA) dilution 1:500 overnight. The polyclonal anti-Tom20 antibodies (Cell Signaling, Danvers, MA, USA) at dilution 1:1000 were used to normalize the protein. Immunoreactivity was determined using secondary antibodies conjugated with horseradish peroxidase (Jackson Immuno Research, West Grove, PA, USA). Peroxidase activity was determined with an ECL (Bio-Rad, Hercules, CA, USA) using a ChemiDoc Touch imaging system (Bio-Rad, Hercules, CA, USA). Protein bands were quantified by densitometry (Image Lab software, Bio-Rad, Hercules, CA, USA) as the ratio of proteins to Tom20.

Cells were grown on coverslips in 12 or 24-well plates. Following treatment, cells were fixed using 4% formaldehyde and permeabilized using 0.2% Triton X-100. After blocking with 4% BSA, cells were incubated with anti-Tom 20 antibody (Cell signaling) and Phalloidin-Alexa488 (Invitrogen), followed by the Cy3-conjugated anti-rabbit IgG (Jackson Immuno Research or Invitrogen), and observed using a confocal microscope (Zeiss, LSM700 or LSM780). To quantify perinuclear mitochondria, the ratio of Cy3 fluorescence intensity of the region surrounding nucleus region (within 5 μm) to the total intracellular Cy3 fluorescence intensity was calculated using ZEN microscopy software.