Fusion pulse 6

The signal of the above-mentioned primary goat anti-human TRPC4 was confirmed via Western blot (Supplementary Figure S1). Proteins were separated using a 10% Mini-Protean^® TGX precast gel (BioRad, Hercules, CA, USA). After blotting, PVDF membranes were blocked with 5% skimmed milk in TBS buffer with 0.1% Tween for 1 h at room temperature and incubated with the primary antibody overnight. Incubation with the secondary antibody was performed for 1 h at room temperature. Blots were analyzed using the chemiluminescence system Fusion Pulse 6 (Vilber Lourmat, Collégien, France). For the detection of multiple antigens, antibodies were removed by incubation with the re-blot mild stripping solution (Merck, Rahway, NJ, USA) for 15 min. Primary antibodies: goat anti-TRCP4 (Thermo Fisher Scientific, Waltham, MA, USA), rabbit anti-actin (Sigma-Aldrich); secondary antibodies: rabbit anti-goat, goat anti-rabbit (both Agilent/Dako, Santa Clara, CA, USA).

Cell extract preparation was performed as described previously (Bienholz et al. 2017 (link)). The shown proteins were visualised using a rabbit antibody against Phospho-Drp1-S637 (Cell Signaling, Danvers, Massachusetts, USA, #4867), Phospho-Drp1-S616 (Cell Signaling, #4494), Drp1 (Cell Signaling, #5391), Opa1 (Cell Signaling, #80471), Fis1 (Thermo Scientific, Waltham, Massachusetts, USA, #PA5-22142), Mfn1 (Merck, Darmstadt, Germany, #ABC41), Caspase-3 (Cell Signaling, #9662), Mff (Cell Signaling, #84580), Phospho-Mff (Cell Signaling, #49281), MiD51 (Proteintech, Rosemont, Illinois, USA, 20164-1-AP), MiD49 (Proteintech, 28718-1-AP) and a mouse antibody against Oma1 (Santa Cruz, Dallas, Texas, USA, sc-515788) and Mfn2 (Abcam, Cambridge, United Kingdom, #ab56889), all in 1:1000 dilution. Anti-rabbit IgG (Sigma-Aldrich, St. Louis, Missouri, USA, #A8275) and anti-mouse IgG (Sigma-Aldrich, #A4416) conjugated with peroxidase was used as secondary antibody in 1:5000 dilution. The chemiluminescence signals (SuperSignal™ West Femto Maximum Sensitivity Substrate, Thermo Fisher Scientific, Waltham, Massachusetts, USA) were recorded using the gel documentation system Fusion Pulse 6 (Vilber Lourmat, Eberhardzell, Germany) and quantified by the software Bio1D (Vilber Lourmat, Eberhardzell, Germany). Gels stained with Coomassie were used as a loading control.

HGFs were trypsinized and lysed with a protein extraction reagent. The concentration of protein was recorded sequentially using the Bradford Kit (Beyotime) and the microplate spectrophotometer (Bio-Tek, Epoch 2) at 595 nm wavelength. Proteins (20 μg) were loaded and then electro-transferred to a polyvinylidene difluoride (PVDF) membrane, which was incubated subsequently with specific primary antibodies as previously described (Wang X. et al., 2016 (link)). Ultimately, the protein bands were quantified using the imaging system (Vilber, Fusion Pulse 6).