Any kd mini protean tgx precast protein gel

The lyophilized products were freshly reconstituted with ddH₂O. A total of 15 µg (based on the manufacturer’s specifications) of venom were applied under reducing conditions to Any kD™ Mini-PROTEAN^® TGX™ Precast Protein Gels (Bio-Rad, Munich, Germany) and proteins separated according to their molecular weight. Bands were visualized using SyproRuby (Thermo Fisher Scientific, Waltham, MA, USA) staining and Typhoon scan (350–400 PMT/25 µm) (Cytiva, Marlborough, MA, USA). Samples were loaded and ran on a single SDS-PAGE.

For in vivo crosslinking experiments, M. xanthus cells were starved for 6 h in MC7 buffer in suspension in the presence of PI at a calculated density of 10⁹ cells per ml. Cells were pelleted at 15,000 × g for 10 min at RT and washed in PBS. After a second centrifugation step under the same conditions, cells were resuspended in the same volume of PBS. Where indicated, DMSO or the crosslinking agent DSP (Thermo Fischer Scientific) resuspended in DMSO to 0.25 mM final concentration were added and cells were incubated for 30 min at 30 °C with gentle agitation. The crosslinking reaction was quenched with 50 mM Tris-HCl (pH 7.6) for 15 min at RT, and cells were pelleted at 15,000 × g for 10 min at RT. Cell pellets were resuspended in 1/10 of the volume of PBS, sonicated, and centrifuged at 5000 g for 10 min at 4 °C to remove cell debris. Supernatants were filtered through a 0.22 µm sterile filter and ultracentrifuged at 40,000 × g for 1 h at 4 °C. Pellets containing membrane fraction (washed once more in PBS) were resuspended in loading buffer (62.5 mM Tris-HCl (pH 7.6), 4% SDS, 20% glycerol, and 0.25% bromophenol blue) with or without 50 mM DTT and separated in Any kD™ Mini-PROTEAN^® TGX™ Precast Protein Gels (Bio-Rad). Proteins were detected by immunoblotting using α-PopC and α-Oar antibodies^{7 (link),51 (link)}.

Tetracycline-inducible Mfn2 and Drp1 knockout tumors were lysed using T-PER Tissue Protein Extraction Reagent from Thermo Fisher Scientific (Waltham, MA, USA). Similarly, KPC cell lines treated with leflunomide were lysed using M-PER Mammalian Protein Extraction Reagent from Thermo Fisher Scientific. Lysates were run on Any kD mini-protean TGX Pre-cast protein gels from Bio-Rad (Hercules, CA, USA) and transferred onto PVDF with a Bio-Rad Trans-Blot Turbo transfer system as previously described [3 (link),11 (link)]. Blots to probe for Mfn2 and Drp1 were run concurrently in the same apparatus in order to simultaneously blot for both proteins. Vinculin was used as the loading control. Primary antibodies against Vinculin, Mfn2, and Drp1 were purchased from Cell Signaling Technology (Beverly, MA, USA) [3 (link)]. HRP-conjugated secondary antibodies were from Thermo Fisher Scientific to probe for primary antibodies. We used the Pierce™ ECL Western Blotting Substrate from Thermo Fisher Scientific (Waltham, MA, USA) for chemiluminescence detection on a ChemiDoc imaging system by Bio-Rad (Hercules, CA, USA).