Las 3000 mini digital imaging system

Cytoplasmic extracts were obtained as previously described (16 (link)). Briefly, Neuro-2a cells (1x10⁷ cells) were resuspended in 1 ml of RIPA buffer (cat. no. 08714-04; Nakalai Tesque, Inc.) at 4˚C for 30 min, and then the cells were centrifuged (8,000 x g) at 4˚C for 15 min. The proteins (40 µg/lane), quantified using bicinchoninic acid assay (BCA), were electrophoresed on a 10% sodium dodecyl sulfate polyacrylamide gel followed by semi-dry transfer to a polyvinylidence fluoride (PVDF) membrane (cat. no. 88518; Invitrogen; Thermo Fisher Scientific, Inc.). The transferred PVDF blots were pretreated with 5% nonfat dry milk in Tris-buffered saline containing 0.1% Tween-20 (TBST) for 1 h at room temperature and incubated with primary antibodies for nuclear factor-κB (NF-κB; product no. 8242; 1:3,000; Cell Signaling Technology, Inc.) and actin (cat. no. A2066; 1:3,000; Sigma-Aldrich; Merck KGaA) at 4˚C overnight. The membranes were then washed three times with TBST and incubated with horseradish peroxidase-conjugated secondary antibody (product code ab99697; 1:3,000; Abcam) for 1 h at room temperature. Following washing three times again, the antibodies bound to the protein blots were detected using Western Lightning Plus Chemiluminescence Reagent (cat. no. NEL103E001EA; PerkinElmer Life Sciences), visualized on a LAS-3000 Mini Digital Imaging System (FUJIFILM Corporation).

Retinal proteins (60 μg) from whole-cell homogenates (4 eyes per group at 4 weeks after treatments) or eluates of the above-mentioned Dynabeads-antibody-antigen complexes were incubated in Laemmli loading buffer containing 20 mM dithiothreitol at 95°C for 5 min, followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (Thermo Fisher Scientific KK) and subsequent transfer on a polyvinylidene difluoride membrane (GE Healthcare Life Sciences, UK). The membrane was blocked with 5% BSA in Tris-buffered saline containing 0.1% Tween 20 (TBS-T) for 1 h at room temperature, then incubated overnight at 4°C with primary antibodies in TBS-T, including rabbit anti-AQP9 (1:5,000, Abcam), MCT1, MCT2, and MCT4 (1:1,000, see above), and β-actin (1:200, Abcam). Then, the membrane was incubated with horseradish peroxidase-conjugated anti-rabbit IgG (1:2,000) at room temperature for 1 h. ECL reagents (GE Healthcare Life Sciences) was used for chemiluminescence detection. The relative ratio of signal to β-actin expression was quantified for total protein immunoblots, while the relative ratio of signal to WT control was quantified for immunoprecipitate immunoblots, using a LAS-3000 Mini digital imaging system (Fujifilm, Tokyo, Japan).