Instantblue coomassie dye

To quantify Drp1 oligomerization, a sedimentation assay was conducted similar to what has been described previously^{9 (link), 53 (link)}. Large oligomers formed by Drp1 samples, in the presence of ligands, were found in the pellet after a medium speed centrifugation. Specifically, protein was diluted in PBS buffer to 0.05 mg/ml (0.62 µM), and specified WT and ∆VD mutant samples were incubated at room temperature with lipid nanotubes (200 µM) and/or GMPPCP (1 mM) for at least 30 minutes. The mixtures were then spun at 13,200 rpm (16,100 × g) for 30 min in a tabletop centrifuge (Eppendorf). The supernatant and pellet fractions were separated, collected, and immediately mixed with SDS-PAGE loading dye (Bio-Rad) and heated briefly at 100 °C. These samples were run on an SDS-PAGE gel and stained with an InstantBlue Coomassie dye (Expedeon). Gels were scanned (HP Scanjet 8300) and densitometry analysis was done using the ImageJ software^{54 (link)}.

To quantify Drp1 oligomerization, a sedimentation assay was conducted similar to what has been described previously^{34 (link),35 (link)}. Large oligomers formed by Drp1 samples, in the presence of ligands, were found in the pellet after a medium speed centrifugation. Specifically, protein was diluted in HEPES KCl buffer to 2 μM, and specified WT and mutant samples were incubated at room temperature with lipid nanotubes (150 μM) and/or GMPPCP (2 mM) for at least 60 min. The mixtures were then spun at 15,000 rpm for 10 min in a tabletop centrifuge (Eppendorf). The supernatant and pellet fractions were separated, collected, and immediately mixed with SDS-PAGE loading dye (Bio-Rad) and heated briefly at 100 °C. These samples were run on an SDS-PAGE gel and stained with an InstantBlue Coomassie dye (Expedeon). Gels were scanned using an Odyssey XF Imaging System (Li-Cor) and densitometry analysis was done using the Image Studio Lite Ver 5.2.

Bacteria were grown overnight under T3SS inducing conditions in LB media containing 5 mM ethylene glycol-bis(2-aminoethylether) (EGTA) (Sigma-Aldrich, St. Louis, MO, USA). The next day the suspension was diluted, and compounds were added to a final concentration of 50 μM in a 50 mL tube. After another 4 h of co-incubation the supernatant was separated via centrifugation and filtered through a 0.45 μm-pore-size low protein-binding filter (ThermoFisher, Scientific, Waltham, MA, USA). Subsequently proteins were precipitated by tricholoroacetic acid (Sigma Aldrich, St. Louis, MO, USA), washed and analyzed by SDS-PAGE using Instant Blue Coomassie dye (Expedeon, Heidelberg, Germany).