Anti akr1c3 clone np6 g6 a6

Cells and spheroids were harvested as previously described [11 (link)]. Whole cell lysates were generated using Tris Glycine SDS sample buffer (Gradipore) by shaking at room temperature for 1 h and further processed via SDS-PAGE as described previously [16 (link)]. The following primary antibodies were used: anti-AKR1C3 (clone NP6.G6.A6, 1:500, Sigma), anti-HMGCS2 (mitochondrial) (ab137043, 1:300, Abcam), and anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH, 1:50000, Millipore). Visualization and quantification of protein bands were performed with Image Studio software Version 5.2 (LI-COR Biosciences).

Cells were lysed using RIPA buffer supplemented with 0.5 mM PMSF, 2 µg/mL leupeptin, 2 µg/mL aprotinin, 2.5 mM NaF and 1% Triton X-100 by shaking at 4 °C for 1 h. Fifty micrograms of protein were separated using 4–12% NuPAGE Bis-Tris protein gels (Thermo Fisher ScientificTM, Vienna, Austria) and transferred onto a 0.2 µm nitrocellulose membrane (GE Healthcare, Vienna, Austria). Blocking of membranes and antibody incubation was performed in StartingBlockTM (PBS) blocking buffer (Thermo Fisher ScientificTM, Vienna, Austria) using the following antibodies: anti-AKR1C3 (clone NP6.G6.A6, 1:500, Sigma), anti-androgen receptor (1:500, Cell Signaling), anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH, 1:50000, Millipore). Visualization and quantification of protein bands were performed with Image Studio software Version 5.2 (LI-COR Biosciences).