Las3000 mini lumino image analyzer

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]).

Harvested cells were lysed with RIPA lysis buffer. Cell lysates or samples prepared with immunoprecipitated beads were separated by SDS-PAGE and transferred onto a polyvinylidene difluoride (PVDF) membrane using a Trans-Blot semidry transfer cell (Bio-Rad, Seoul, South Korea). Then, the membrane was blocked for 1 h in 5% bovine serum albumin (BSA) and incubated overnight at 4°C with the primary antibody. The next day, membranes were washed with Tris-buffered saline with Tween 20 (TBST) or phosphate-buffered saline with Tween 20 (PBST), and the membrane was incubated with horseradish peroxidase (HRP)-conjugated secondary antibody for 2 h at room temperature. The membrane was washed again 3 times with TBST or PBST, and finally, the reaction was visualized using an enhanced chemiluminescence (ECL) detection system (GE Healthcare, Little Chalfont, United Kingdom) using a Las-3000 Mini Lumino image analyzer.