V5 hrp antibody

The level of expressed microsomal SDR protein in each over-expressed cell line was determined by western blot analysis using a monoclonal V5-HRP antibody (Invitrogen; Thermo Fisher) at a 1:5000 dilution as recommended by the manufacturer. Microsomal protein (0.7–30 µg) from each SDR-containing cell line was loaded into each lane. The monoclonal Calnexin-HRP antibody (Sigma-Aldrich) at a 1:5000 dilution was used as a loading control. The intensity of V5 signal was measured using Image J (National Institutes of Health, Bethesda, MD). The relative V5-protein expression of each SDR was calculated as the mean of three independent experiments and relative to the V5 signal for the HSD11β1-over-expressing cell line. All activity assays were normalized based on the relative V5-protein expression within each respective SDR-over-expressing cell line, with the V5-protein expression equal to 1.9, 0.9, 1.3, 4.3, 0.4, 0.4 and 0.2 for cell lines over-expressing HSD11β1, HSD17β6, HSD17β12, KDSR, NSDHL, RDH10, RDH11 and SDR16C5, respectively, as compared to that observed for HSD11β1 (set to 1.0).

For SDS-electrophoresis, proteins isolated as crude membranes as described above, were separated using 4–15% precast gradient TGX Stain-Free gels (Bio-Rad, Hercules, CA) for 35 min at 200 V. Proteins were transferred to polyvinylidene difluoride membranes using the Trans-Blot Turbo Transfer System (Bio-Rad). Membranes were blocked with 5% blotting-grade blocker (Bio-Rad) in Tween-Tris-buffered saline (TTBS, 100 mM Tris (pH 7.5), 0.9% NaCl, 1% Tween-20 [25–30 mL/membrane]) overnight. The membranes were incubated with the primary V5 horseradish peroxidase (HRP)-conjugated antibody (1:5000, V5-HRP antibody; Invitrogen, Carlsbad, CA) in TTBS with 5% blotting-grade blocker overnight at 4°C. After the removal of the primary antibody, the membranes were washed five times for 5 min with TTBS. Proteins were visualized by chemiluminescent detection (ImageQuant LAS 4000; GE Healthcare Life Sciences, Chicago, IL) of peroxidase substrate activity (SuperSignal West Femto Maximum Sensitivity Substrate; Thermo Fisher Scientific). Band intensities were quantified using UN-SCAN-IT gel (Silk Scientific, Orem, UT). The DTT-resistant band (running at ∼130 kDa) was excluded from the quantification but caused the background of the membrane to appear darker in close proximity to the location of the dimer, potentially increasing the band intensity.