Irdye 680 800cw

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.

Protein lysates were prepared in cell lysis buffer (Cell Signaling Technology, Denver, CO, USA) and separated via sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS–PAGE) before being transferred to FL-PVDF membranes. Blots were incubated overnight at 4 °C using primary antibodies (Table S3). Next, the membrane was washed with TBST and subjected to fluorescence-conjugated secondary antibodies (LI-COR IRDye 680/800 CW, 1:15,000 dilution). Fluorescence visualization was performed using an LI-COR Odyssey CLx Imager (Lincoln, NE, USA).