Immobilon western detection system

Equal volumes (10 μl) of representative fractions from OptiPrep gradients were separated by SDS-PAGE (10% polyacrylamide gel), and the proteins were transferred to a Hybond-ECL filter (Amersham Bio-sciences). The filter was equilibrated in TTBS buffer (25 mM Tris, 150 mM NaCl, and 0.1% Tween-20) for 10 min and incubated in blocking buffer (5% w/v milk in TTBS) for 1 h at room temperature. Monoclonal antibodies against HA or 3XFLAG epitope were added at a dilution of 1:10,000 and incubated for 1 h at room temperature. The bound antibody was detected by using anti-mouse IgG antibody conjugated to horseradish peroxidase (Sigma Aldrich) and the Immobilon Western detection system (Millipore). It has to be mentioned that immunodetection of HflC-HA and YbbK-3Flag was carried out on all independently generated membrane fractions, including the ones used for proteomics. Immunodetection of all other protein markers was carried out on at least three independently generated membrane fractions, but not the ones used for proteomic analysis.

The cultures grown aerobically in LB were harvested during midexponential growth, and the cells were washed with Tris/HCl buffer (50 mM Tris/HCl pH 7.6, 200 mM KCl, 10 mM MgCl2, and 0.1 mM EDTA). The cell pellet was resuspended in 5 ml of the same buffer and disrupted by sonication. Cell debris was removed by centrifugation for 10 min at 10,000g. The supernatant was centrifuged for 45 min at 30,000g to separate the cytosolic and the membrane fractions. The resultant supernatant fluid containing the soluble proteins was collected. The remaining pellet was resuspended in 0.5 ml of Tris/HCl buffer. The samples of cytosolic and membrane (containing 5 μg of protein) fractions were separated by SDS-PAGE (10% polyacryl-amide gel), and the proteins were transferred to a Hybond-ECL filter (Amersham Biosciences). The filter was equilibrated in TTBS buffer (25 mM Tris, 150 mM NaCl, and 0.05% Tween 20) for 10 min and incubated in blocking buffer (1% milk in TTBS) for 1 h at room temperature. BarA polyclonal antibodies, raised against His6-BarA^198–918 (33 (link)), were added at a dilution of 1:10,000 and incubated for 1 h at room temperature. The bound antibody was detected by using anti-rabbit IgG antibody conjugated to horseradish peroxidase and the Immobilon Western detection system (Millipore). The protein bands were quantified using ImageJ software (61 (link)).

Cells were lysed in the RIPA buffer supplemented with cocktails of protease/phosphatase inhibitors (Cell Signaling Technology, Beverly, MA, USA). Proteins (20 μg) were separated using a 10% SDS–polyacrylamide gel and the Bio-Rad electroporation system and then transferred onto PVDF membranes (Millipore, Billerica, MA, USA). CD46 antibodies were obtained from OriGene Technologies, Inc. (TA306994, Rockville, MD, USA).
CD55 (sc-51733), CD59 (sc-133170), p-AKT (sc-514032), AKT (sc-5298), p-ERK1/2 (sc-136521), and ERK1/2 (sc-514302) were procured from Santa Cruz Biotechnology (SantaCruz, CA, USA). β-Actin antibodies were obtained from Sigma-Aldrich (A5441, St. Louis, MO, USA). The bands were visualized and analyzed using the Immobilon Western detection system (Millipore, Billerica, MA, USA) and ChemiDOC™ MP Gel Imaging System (Bio-Rad, Hercules, CA, USA).