Image studio digits 5

Cells were lysed in a buffer containing 4% sodium dodecyl sulfate (SDS), 150 mM NaCl, and 100 mM Tris/HCl, pH 7.4, and sonicated. Protein content was determined by a protein assay kit (Bio-Rad, Munich, Germany), and 100 µg protein was loaded onto a 10% SDS gel. Gels were blotted by using a Trans Blot Turbo blotting system (Bio-Rad).
Membranes were blocked in 5% milk in Tris-buffered saline with 0.05% tween 20 (TBS-T) for tubulin (Sigma-Aldrich, Munich, Germany), or 5% bovine serum albumin (BSA) in TBS-T for TMEM126B (Atlas Antibodies via Sigma-Aldrich, Munich, Germany), electron transferring flavoprotein-ubiquinone oxidoreductase (ETFDH) (Abcam, Berlin, Germany), and oxidative phosphorylation (OXPHOS) Western cocktail (abcam). Enhanced chemiluminescence on a C-DIGIT scanner (Licor, Lincoln, USA), or fluorescence on an Odyssey scanner (Licor), were quantified with Image Studio Digits 5.0 (Licor).

Cells were lysed in a buffer containing 6.65 M urea, 10% glycerol, 1% SDS, 10 mM Tris/HCl, pH 7.4 and sonicated. Protein content was determined by a protein assay kit (Bio-Rad, Munich, Germany) and 60 µg protein was loaded on a 10% SDS gel. For HIF 7.5% SDS gels were used. Gels were blotted using a Trans Blot Turbo blotting system (Bio-Rad). Before blocking membranes were stained using the Revert™ 700 Total Protein Stain kit (Licor, Lincoln, USA) according to manufacturer’s instructions. Afterwards, membranes were blocked in 5% milk in TBS-T for Akt (9272, Cell Signaling, Frankfurt, Germany), pAkt Thr 308 (5106, Cell Signaling), pAkt Ser 473 (4051, Cell Signaling), PPTC7 (HPA039335, Sigma-Aldrich), HIF-1α (610959, BD, Heidelberg, Germany), PFKFB2 (39527, Cell Signaling), PFKFB2 Ser 483 (39527, Cell Signaling), PFKFB3 (ab181861, abcam, Cambridge, UK), and PFKFB3 Ser 461 (ab202291, abcam). Fluorescence signal was detected on an Odyssey scanner (Licor) and quantified with Image Studio Digits 5.0 (Licor). For each lane the lane normalization factor (LNF) was calculated (intensity of a complete lane divided by the intensity of the lane with the maximal intensity) and used for normalization of the signal of the corresponding primary antibody. Complete total protein stains are collectively shown in Figure S1.