Immunodetection of Hsd3b6 using Blue Native PAGE

The antibody that we developed for Hsd3b6 (ref. ^{21 (link)}) could not be used for SDS–PAGE-based blotting^{21 (link)}. We thus employed Coomassie brilliant blue-based blue native PAGE for immunodetection of Hsd3b6. Meibomian glands were homogenized in 50 mM Tris-HCl (pH 7.2) buffer containing 1 mM dithiothreitol and 1× cOmplete Protease inhibitor cocktail (Roche diagnostics), followed by centrifugation at 106,000g. The pellet was resuspended in NativePAGE Sample Buffer (Invitrogen) containing 1 mM dithiothreitol and 1% digitonin and mixed with NativePAGE 5% G-250 Sample Additive (Invitrogen) before loading on the NativePAGE Novex Bis-Tris Gel (Invitrogen). Electrophoresis was performed at a constant current of 1 mA at 4 °C. Proteins in the gel were electroblotted to a polyvinylidene difluoride membrane with a buffer containing 25 mM Tris, 200 mM glycine, 10% methanol and NativePAGE Cathode Additive (Invitrogen) at a constant current of 350 mA for 1.5 h. Membranes were incubated further with PBS containing 0.05% Tween-20 for 24 h to remove Coomassie brilliant blue on the blots. After incubation with a Blocking One buffer (Nacalai), blots were probed for Hsd3b6 (anti-Hsd3b6) and its immunoreactivities were visualized using chemiluminescence. Immunoblot for Per2 was performed as described^{38 (link)}. Full-length western blots are presented in Supplementary Information.