Protein extraction and immunoblotting

Cells were lysed using RIPA buffer (Sigma-Aldrich), supplemented with protease and phosphatase inhibitor cocktails (both 1/100) (Thermo Fisher Scientific). Protein concentrations were determined using the Bio-Rad protein assay (Bio-Rad Laboratories Inc.), according to the manufacturer’s instructions. Primary human AKR1D1 (dilution 1/250 – HPA057002, Atlas Antibodies AB, Bromma, Sweden), β-tubulin (#15115, monoclonal) (Cell Signalling), β-actin (#3700, monoclonal) (Cell Signalling), and secondary antibodies (P044801-2, polyclonal) from Dako (Agilent) were used at a dilution 1/1000 (primary) and 1/2000 (secondary), respectively, unless stated otherwise. Bands were visualised with Bio Rad Clarity Western ECL (Watford, Hertfordshire, UK) and ChemiDocXS imager (Bio Rad). Western blots were quantified by densitometry analysis using ImageJ (NIH, http://rsb.info.nih.gov/ij), normalised to β-tubulin and β-actin to correct for variability in gel loading.