Protein assay system

Proteins were extracted using RIPA Lysis buffer, which volume was adapted for every tissue. Proteins extracted were quantified using Bio-Rad Protein Assay System (Bio-Rad Laboratory, Melville, NY). Protein lysates were boiled in Laemmli buffer, run on BIO-RAD Mini-Protean TGXTM (10–20% Ready Gel Tris-HCl Gel System, 12-well comb #456–1095 and 10-well comb #456–1094) for 90 min at 120 Volt, and transferred on BIO-RAD Trans-Blot Turbo Mini or Midi PVDF membranes (Mini PVDF Transfer Packs #170–4156 and Midi PVDF Transfer Packs #170–4159) by semi dry Transfer (Trans-Blot Turbo Transfer System BIO-RAD). Specific proteins were detected using the following primary antibodies: Connexin-43 (1:850) (Cell Signalling, #3512, RRID:AB_2294590), β-actin HRP-conjugated (1:5000) (Sigma-Aldrich, #2228, RRID:AB_476697). Antibodies were diluted in 5% milk and incubation was done overnight at 4°C. Following secondary antibodies were used: HRP-conjugated anti-rabbit (1:5000) (Dako, #P0448, RRID:AB_2617138). Membranes were then washed and protein expression was analysed by chemiluminescence (Western Lightning Plus-ECL Perkin Elmer), using Fusion-FX (Vilber).

Brain lysates were prepared from P37 male mouse forebrains (2 WT mice and 2 KO mice, independently) with a RIPA buffer (20mM HEPES pH 7.4, 100mM NaCl, 1mM EDTA, 1% Triton X-100 containing Protease Inhibitor cocktail (Nacalai, Kyoto, Japan)) using a probe sonicator (TOMY UD-201). The protein concentration of the brain lysate was quantified by BioRad Protein Assay System (BioRad, Hercules, USA). Protein in the amount of 0.1mg was subjected to SDS-PAGE (7.5% LaemmLi) and electro-blotted onto Immobilon-FL PVDF membrane (Millipore, Burlington, USA). Western blotting was carried out using anti-IQSEC2 rabbit polyclonal antibody (PAS72831, 1/2000 dilution, Thermo Fisher Scientific,Waltham , USA) and anti-β-Actin mouse monoclonal antibody (MBL, M177-3, 1/5000 dilution), as primary antibodies. IRDye 800CW Goat anti-Rabbit secondary antibody (LI-CDR, 1/5000 dilution) and IRDye 800 Goat anti-Mouse secondary antibody (LI-CDR, 1/5000 dilution) were used as secondary antibodies. Infrared signals were detected by ODYSSEY Imaging System (LI-CDR Bioscience, Lincoln, NE, USA).

Cultured CG cells were solubilized with RIPA buffer (20 mM HEPES pH 7.4, 100 mM NaCl, 1 mM EDTA, 1% Triton-X 100 containing the protease inhibitor cocktail (Nacalai tesque, Kyoto, Japan). The protein concentration of the CG cell lysate was quantified by the BioRad Protein Assay System (BioRad, Berkeley, CA, USA). Twenty μg protein was subjected to SDS-PAGE (7.5% Laemmli) and electroblotted onto Immobilon-FL PVDF membrane (Millipore, Burlington, MA, USA). Western blot was performed using anti-Flag mouse monoclonal antibody (MBL, M185-3S, 1:1000), anti-CASK polyclonal antibody (Abcam, Cambridge, UK, ab3383, 1:1000), anti-CASK monoclonal antibody (NeuroMab, K56A/50, 1:1000), anti-β-Actin mouse anti-Actin monoclonal antibody (MBL, Tokyo, Japan, M177-3, 1:1000), IRDye 800 CW goat anti-rabbit secondary antibody (LI-CDR Bioscience, Lincoln, NE, USA, 1:20,000), IRDye 800 goat anti-Mouse secondary antibody (LI-CDR Bioscience, Lincoln, NE, USA, 1:20,000) and visualized by ODYSSEY Imaging System (LI-CDR Bioscience, Lincoln, NE, USA).