Semi dry turbo transfer system

Lysates were prepared from clonal populations of cells in a proteolysis inhibitor (Sigma) before solution in 2x reducing buffer. Western Blots were resolved by SDS-PAGE on 4–12% Bis/Tris gradient NuPage gels (Thermo Scientific). Proteins were transferred to polyvinylidene difluoride (PVDF; Bio-Rad) membranes using a Bio-Rad semi-dry Turbo transfer system. Membranes were blocked with 4% BSA in 0.1% Tween-20 Tris-buffered saline (TBST; 200 mM Trizma base, 1.37 M NaCl, 0.1% Tween-20) and probed with antibodies to α-tubulin (Sigma), GAPDH (loading control) (Abcam) and after antibody stripping with antibodies to eGFP (Sigma) and α-tubulin (Sigma). For quantification of TubA1B expression, wild type lysates (Hek293T, Hel 92.1.7, A549) were first probed with a TubA1B antibody (Abcam) and a β-actin loading control before stripping and re-probing with antibodies to α-tubulin (Sigma) and GAPDH (loading control). Values were normalized to loading controls, and then normalized to brightness to give relative intensity values. All primary antibodies were diluted in the blocking buffer shown above. Secondary incubations were performed in anti-mouse 680 and anti-rabbit 800 fluorescent antibodies (LiCor Instruments) for fluorescent detection using an Odyssey Fc (LiCor Instruments).

HR-2 cells were grown in DMEM-S in 10-cm dishes for 24 h with 0.20 mM oxygen before they were moved to lower oxygen concentrations (0.04, 0.02, and 0.005 mM) for 48 h before harvest. At the time of harvest, cells were scraped into radioimmunoprecipitation lysis buffer (Thermo Fisher Scientific, Waltham, MA, USA) and sonicated. Protein levels in the lysates were quantified by BCA (Thermo Fisher Scientific). Western blot analysis was conducted with an electrophoresis chamber and semi-dry turbo transfer system (BioRad, Hercules, CA, USA). The antibodies used were: LDHA: mAB #3582, P-LDHA pAB #8176, HSP90 (mAB #4877) (Cell Signaling Technology, Danvers, MA, USA); MCT-1 (pAB: T-19, sc-14917) and MCT-4 (pAB: H-90, sc-50329) (Santa Cruz Biotechnology, Dallas, TX, USA).