Transketolase

Cells and tumour tissue were lysed in RIPA buffer (50 mM Tris, pH 7.5, 150 mM NaCl, 1% NP40, 0.5% sodium deoxycholate, 0.1% SDS, 2 mM sodium orthovanadate, 50 mM NaFl, 1 mM sodium molybdate, 40 mM β-glycero-phosphate, 1 mM PMSF and 1/100 protease inhibitor cocktail) and protein concentration of lysates was determined using DC Protein Assay Kit (Bio-Rad). A total of 25 μg of protein was subjected to gel electrophoresis on a 10% SDS-PAGE gel and transferred to a PVDF membrane for one hour at 100 V. Membranes were washed in TBST (Tris-buffered saline with 0.2% Tween 20) and blocked with 5% skim-milk/TBST. Membranes were incubated at 4 °C overnight with the following primary antibodies: G6PD (12263, Cell Signaling), PSAT1 (ab96136, Abcam), PHGDH (ab13428, Abcam), transketolase (8616, Cell Signalling), transaldolase (PA5-27614, ThermoFisher) and β-actin (A2228, Sigma Aldrich), Membranes were washed in TBST and incubated with their respective HRP-conjugated secondary antibodies (Sigma) in 5% skim-milk/TBST, washed with TBST and visualised using SuperSignal West Pico PLUS Chemiluminescent Substrate kit (34577, ThermoFisher). Original blot images can be found in Supplementary File 1. Densitometry analysis was performed using ImageJ and normalised to β-actin expression to determine the adjusted density value for each protein.