Blocking one buffer

Cell culture was the same as previously described. Whole-cell lysates were prepared for western blotting as we previously performed [22 ]. The samples (2 μg for ABCB1 measurement, 30 μg for others) were equally subjected to 4–15% Mini-PROTEAN TGX Precast Protein Gels (Bio-Rad, Hercules, California, USA) and transferred to Immun-Blot PVDF Membrane (Bio-Rad). After blocking with Blocking One buffer (Nacalai Tesque, Inc., Kyoto, Japan) for 2 h, the membranes were incubated with the indicated antibodies for overnight at 4°C, followed by incubation with horseradish peroxidase-conjugated secondary antibody for 2 h at room temperature. The bands were further dealt with Chemi-Lumi One L solution (NACALAI) and analyzed. The primary antibodies for caspase-8 were obtained from Medical & Biological Laboratories CO., LTD. (Nagoya, Japan) and those for caspase-3, caspase-9, survivin, NF-kappa B p65, p-NF-kappa B p65, Bcl-xL, and Bcl-2-associated X protein (Bax) were from Cell Signaling Technology, Inc. (Danvers, Massachusetts, USA). Primary antibodies for ABCB1 and GAPDH and secondary antibodies goat anti-rabbit IgG H&L (HRP) and rabbit anti-mouse IgG H&L (HRP) were purchased from Abcam (Cambridge, Massachusetts, USA). The density of bands was measured using a LAS4000 fluorescence image analysis system (FUJIFILM, Tokyo, Japan). All experiments were performed three times.

Protein (5–20 μg) from each sample was subjected to SDS-PAGE on 7–8% acrylamide gels. The individual proteins were separated using SDS-PAGE. After SDS-PAGE, the proteins were transferred from the gel onto polyvinylidene difluoride membranes (Immobilon-FL, Millipore, Bedford, MA, USA) using a Trans-Blot Turbo system. Next, the Blocking One buffer (Nacalai Tesque, Kyoto, Japan) was used to block the membranes at room temperature for 30 min. Depending on the primary antibody, the membranes were incubated overnight at 4 °C or at room temperature for 1 h. Anti-PSD-93 (1:1000), anti-phospho-PSD-93 (T612) (1:100), anti-PSD-95 (1:1000), anti-phospho-MYPT1 (T853) (1:1000), anti-MYPT1 (1:1000), anti-c-Myc (1:1000), anti-EGFP (1:1000), anti-GST (1:1000), anti-phospho-CaMKII (T286) (1:1000), anti-CaMKII (1:1000), anti-SynGAP1(1:1000), anti-GluR1 (1:1000), anti-NMDAR1 (1:1000), anti-ADAM22 (1:1000), and anti-LGI1 (1:1000) were used. The unbound primary antibodies were washed away, and the membranes were incubated with goat anti-rabbit Alexa Fluor 680 and/or goat anti-mouse IRDye 800CW for 1 h at room temperature. The total protein or phospho-proteins were detected by infrared imaging (LI-COR Biosciences Lincoln, NE). The band intensities were quantified using ImageStudio software (LI-COR Biosciences).

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.