Chemidocxs imager

Total protein was extracted from cells using RIPA buffer (150 mM NaCl, 1.0%
IGEPAL® CA-630, 0.5% sodium deoxycholate, 0.1% SDS, and 50 mM Tris, pH 8.0)
(Sigma-Aldrich) and protease inhibitor cocktail (Thermo Fisher Scientific). Protein
concentrations were measured using a commercially available assay (Bio-Rad Laboratories)
according to the manufacturer’s protocol. Primary human AKR1D1 (dilution 1/250;
HPA057002, Atlas Antibodies AB, Bromma, Sweden), GILZ (sc-133215, Santa Cruz
Biotechnology), β-tubulin (#15115, monoclonal) (Cell Signaling), β-actin
(#3700, monoclonal) (Cell Signaling), CYP8B1 (#PA5-37088, polyclonal) (ThermoFisher
Scientific) and secondary antibodies (P044801-2, polyclonal) from Dako (Agilent) were used
at a dilution of 1/1000 (primary) and 1/2000 (secondary) respectively, unless stated
otherwise. Bands were visualised with Bio Rad Clarity Western ECL and ChemiDocXS imager
(Bio Rad). Western blots were quantified by densitometry analysis using ImageJ (https://imagej.nih.gov/ij/), normalised to β-tubulin to correct for
variability in gel loading.

Total protein was extracted from cells using RIPA buffer (Sigma-Aldrich, Dorset, UK), protease inhibitor cocktail (1/100) and phosphatase inhibitor cocktail (1/100) (ThermoFisher Scientific, Loughborough, UK). Protein concentrations were measured using a commercially available assay according to the manufacturer's protocol (Bio-Rad, Hemel Hempstead, UK). Primary β-tubulin (#15115, monoclonal), total AKT (#4685, monoclonal), pAKT (#4060, monoclonal), AKT1 (#75692, monoclonal), mTOR (#2983, monoclonal), pGSK3β (#5558, monoclonal), GSK3β (#12456, monoclonal), ACC (#3662, polyclonal), pACC (#11818, monoclonal), IκΒα (#4812, monoclonal) from Cell Signalling (Danvers, USA), AKR1D1 (dilution 1/250) (HPA057002, polyclonal, Atlas Antibodies AB, Bromma, Sweden), and secondary antibodies (P044801–2, P044701–2, polyclonal) from Dako (Agilent Technologies, Santa Clara, USA) were used at a dilution of 1/1000 and 1/2000 respectively, unless stated otherwise. To ascertain equal gel loading, proteins were normalised to β-tubulin. Bands were visualised with ECL and ChemiDocXS imager (Biorad, Hemel Hempstead, UK). Western blots were quantified by densitometry analysis using Image J (NIH, Bethesda, MD, http://rsb.info.nih.gov/ij).

Total protein was extracted from cells using RIPA buffer (150 mmol/L NaCl, 1.0% IGEPAL^® CA‐630, 0.5% sodium deoxycholate, 0.1% SDS, and 50 mmol/L Tris, pH 8.0) (Sigma‐Aldrich, Dorset, UK), and protease inhibitor cocktail (ThermoFisher Scientific, Loughborough, UK). Protein concentrations were measured using a commercially available assay (Bio‐Rad Laboratories Inc., Hercules, CA). Protein levels were determined using the Bio‐Rad assay kit, according to the manufacturer's protocol (Bio‐Rad, Hemel Hempstead, UK). Primary (G6PC [Abcam plc, Cambridge, UK], PEPCK, IRE1, CHOP, β‐tubulin [Cell Signalling, Danvers]) and secondary antibodies from Dako were used at a dilution of 1/1000 and 1/2000, respectively. To ascertain equal gel loading, proteins were normalized to β‐tubulin. Bands were visualized with ECL (Pierce Thermo Fisher Scientific) and ChemiDocXS imager (Bio‐Rad). Western blots were quantified by densitometry analysis using ImageJ (NIH, Bethesda, MD, http://rsb.info.nih.gov/ij).