Mitochondrial Dynamics Regulation in Tumor Cells

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).