Total protein normalization substrate

Adherent cells and spheroids were cultured for 48 h under normoxic and hypoxic conditions. Cells were lysed in radioimmunoprecipitation buffer supplemented with protease and phosphatase inhibitors. Protein concentrations in the lysates were quantified using the Pierce Bicinchoninic acid assay (Thermo Fisher Scientific). Equal protein concentrations were loaded in a 4% stacking and 10% SDS separating gel. Proteins were transferred onto a PVDF membrane (Bio-Rad) and blocked with 5% milk in TBST. Primary antibodies were used against DRP1, OPA1 (Novus Bio), MFN1, FIS1 (Protein Tech), UCP2 (Santa Cruz), UCP3 (ThermoFisher Scientific), TOMM 20 (Millipore), and superoxide dismutase 2 (SOD2) antibody (Cell Signaling). Proteins were normalized using the ribosomal protein L19 (L19) or to total protein normalization substrate (Thermo Fisher Scientific). Blots were subsequently probed with the appropriate HRP-conjugated mouse and rabbit or IRDye 680/800cw (Licor) secondary antibodies Proteins were visualized using chemiluminescence Pico ECL (ThermoFisher Scientific) solution with an exposure of 15–30s using the transilluminator from Bio-Rad or the Licor Odyssey Clx imaging system. Proteins quantified using ImageJ software. Data presented as mean ± SEM from at least three biological replicates.

Adherent spheroids harvested at the indicated timepoints and culture conditions were lysed in radioimmunoprecipitation buffer supplemented with protease and phosphatase inhibitor tablets (ThermoFisher). Protein concentrations were determined using the Pierce Bicinchoninic Acid assay (ThermoFisher), normalized to 1 µg/µL and mixed with 50:1 2x Laemmli sample buffer (BioRad): 2-mercaptoethanol (ThermoFisher). Samples were run in a 10% acrylamide SDS gel and transferred onto a PVDF membrane (BioRad). Total protein was used to normalize individual proteins as was quantified using the total protein normalization substrate (ThermoFisher). Blots were blocked with 5% milk in 1X Tris-buffered saline with 0.1% Tween 20. Primary antibodies against Tomm20 (Millipore), TFAM (Abcam), PGC1α (Millipore), BNIP3 (Abcam), LOX (Abcam), MMP2 (Novus Bio), and MMP9 (Novus Bio), and LC3B (Cell Signaling) were imaged using IRDye 680 cw rabbit and 800 cw mouse secondary antibodies (LiCor) and the Licor Odyssey CLx Imager. Proteins were quantified and normalized to total protein using ImageJ.