Goat anti ldh a

Using published protocols (Perrotta et al., 2015 (link), 2018 (link); Catalani et al., 2016 (link)), cells were homogenized in RIPA lysis buffer, supplemented with a cocktail of protease inhibitors (cOmplete; Roche Diagnostics, Milano, Italy). Equal amounts of proteins were separated by 4–20% SDS-polyacrylamide gel electrophoresis (Criterion TGX Stain-free precast gels and Criterion Cell system; Bio-Rad, Hercules, CA, USA) and transferred onto nitrocellulose membrane using a Bio-Rad Trans-Blot Turbo System. The membranes were probed using the anti-cleaved-caspase 3 primary antibody (Cell Signaling Technology, Danvers, MA, USA). After the incubation with the appropriate horseradish-peroxidase-conjugated secondary antibody (Cell Signaling Technology), bands were visualized using the Clarity Western ECL substrate with a ChemiDoc MP imaging system (Bio-Rad). To monitor for potential artifacts in loading and transfer among samples in different lanes, the blots were routinely treated with the Restore Western Blot Stripping Buffer (ThermoFisher Scientific, Waltham, MA, USA) and re-probed with the goat anti-LDH-A (Santa Cruz Biotechnology, Dallas, TX, USA). Bands were quantified for densitometry using the Bio-Rad Image Lab software.

Cell lysates were separated on 10% SDS-PAGE and transferred to polyvinylidene difluoride membrane. Primary antibodies were used at 1:1,000 unless otherwise stated. The following antibodies were used, mouse anti-HIF1α, (BD Transduction Laboratories, USA), mouse anti-CAIX (Gift form J. Pastrorek, Institute of Virology, Slovak Republic), goat anti-LDHA (Santa-Cruz), goat anti-PDK1 (Santa-Cruz), goat anti-AK4 (Santa-Cruz), mouse anti-ALDH (BD Biosciences), mouse anti-β actin (Sigma). Blots were quantified with image analysis in ImageJ.