Las4000 mini lumino image analyzer

Brains and MEFs were lysed in TNE-N buffer (1% NP-40, 150 mM NaCl, 20 mM Tris HCl, pH 7.5, and 2 mM EDTA). Protein concentration was measured by the Bio-Rad Protein assay kit (Bio-Rad). Extracted proteins were boiled for 5 min with 4× NuPAGE LDS (lithium dodecyl sulphate) sample buffer (Thermo Fisher Scientific). Proteins were separated by NuPAGE sodium dodecyl sulphate (SDS)-polyacrylamide gel electrophoresis (PAGE) Gel System (Thermo Fisher Scientific), electroblotted onto a polyvinylidene difluoride membrane (PVDF; Amersham Hybond-P, GE Healthcare). PVDF membrane was masked with 3% non-fat dry milk in TBST (150 mM NaCl, 0.1% Tween20, 25 mM Tris HCl pH7.5) for 30 min at room temperature, incubated with primary antibodies in Can Get Signal IB enhancer Solution I (TOYOBO) for 12 h at 4°C, washed with TBST and incubated with secondary antibodies in 3% BSA for 1 h in TBST at room temperature. After final washes, proteins were visualized using enhanced chemiluminescence, according to the manufacturer’s instructions. Chemiluminescence signals were detected using LAS4000mini Lumino Image Analyzer (GE Healthcare).

The cells were lysed with RIPA lysis buffer in the presence of the protease and phosphatase inhibitor cocktail (Sigma). Individual proteins in the samples were separated via SDS-PAGE and transferred onto a PVDE membrane (Bio-Rad, Hercules, CA, USA) using a Trans-Blot semi-dry transfer cell (Bio-Rad) with buffer containing 30 mM Tris, 200 mM glycine, and 20% methanol. The transferred membranes were incubated at room temperature for 1 h with 5% bovine serum albumin (BSA) in 1× TBST (Tris-buffered saline containing 0.05% Tween 20) for blocking. Then, the membranes were incubated with the target primary antibody at 4 °C overnight, and following that, they were washed 3 times with 1× PBST or TBST for 10 min each. After this step, the membranes were treated with horseradish peroxidase (HRP)-conjugated secondary antibody for 1 h at room temperature, and then, the washing step was repeated another 3 times. The HRP was visualized using an Enhanced Chemiluminescence Femto kit (LPS solution, Daejeon, Republic of Korea) and a LAS-4000 Mini Lumino Image Analyzer (GE Healthcare Life Sciences, Chicago, IL, USA).

This method was used in Figure 1, Figure 2, Figure 3, Figure 4, S1, S3, and S5. Total cellular proteins were solubilized, separated by SDS-PAGE, and electrotransferred onto a polyvinylidene difluoride membrane (Merck Millipore). The membrane was first blocked with 1% or 5% skim milk in PBST (PBS [136 mM NaCl, 2.7 mM KCl, 4.3 mM Na₂PO₄, 1.5 mM KH₂PO₄] with 0.1% Tween-20) and then incubated with anti-YfgM (1/2000 dilution), anti-PpiD (1/20,000 or 1/50,000 dilution), anti-BamB (1/2000 dilution), anti-His (1/2000 dilution), anti-V.SecD1 (1/2000 dilution), or anti-V.SecD2 (1/2000 dilution) antibodies in the blocking solutions at room temperature overnight. After washing with PBST three times, the membrane was incubated with a HRP-conjugated secondary antibody) (1/5000 dilution; Goat Anti-Rabbit IgG (H + L)-HRP conjugate; Bio-Rad Laboratories, Inc) in PBST at room temperature for 1 h. Proteins were visualized with detection reagents (ECL Western Blotting Detection Reagents [GE Healthcare UK Ltd], ECL Prime Western Blotting Detection Reagents [GE Healthcare], and Chemi-Lumi One [Nacalai Tesque]), and chemiluminescence image analyzers (LAS3000 mini lumino-image analyzer [GE Healthcare], LAS4000 mini lumino-image analyzer [GE Healthcare], and FUSION Solo S [VILBER]).