Enhanced chemiluminescence system

Protein lysates were resolved by polyacrylamide gel electrophoresis (PAGE) and electrotransferred onto an Immobilon-P membrane (Millipore) as described previously [27 (link)]. Mouse monoclonal antibody against V5 epitope (Invitrogen) was used as the primary antibody to detect expression of the V5-tagged recombinant proteins. Following incubation with horseradish peroxidase-conjugated secondary antibodies, specific interactions between antigens and antibodies were detected by the enhanced chemiluminescence system (Advansta).

Recombinant yeast strains expressing different antigens were used in the experiments. The pellet of approximately 10⁷ cells (OD₆₀₀ = 1) was collected 48 h after induction. The pellet was washed thrice with 500 µL of PBS for subsequent analysis using Western blotting, indirect immunofluorescence, and flow cytometry.
For Western blotting, proteins were analyzed by SDS-PAGE and transferred to nitrocellulose membranes. The membranes were blocked with TBST containing 5% skim milk powder and 2% bovine serum albumin (BSA) for 2 h at room temperature. Then, the blots were incubated with anti-V5 monoclonal antibody (Biodragon, Beijing) overnight at 4°C, followed by incubation with horseradish peroxidase (HRP)-labeled goat anti-mouse IgG (Biodragon, Beijing) for 1 h at room temperature. Immunoreactive bands were visualized using an enhanced chemiluminescence system (Advansta, San Jose, CA, USA). For indirect immunofluorescence and flow cytometric analyses, SC cell pellet samples were blocked with 5% BSA at 30°C for 2 h and then incubated with anti-V5 monoclonal antibody at 30°C for 2 h, followed by incubation with DyLight 488-labeled goat anti-mouse IgG (Biodragon, Beijing) for 90 min at 30°C. Then, 5 µL of SC cells were used in immunofluorescence assays (Olympus, Tokyo, Japan), and 300 µL of yeast cells were analyzed by flow cytometry (Agilent, United States).

Cells were lysed in cell lysis buffer (50 mM Tris-HCl, pH7.5, 120 mM NaCl, 1 mM EDTA, and 1% NP-40) containing protease inhibitor cocktail (Roche, Indianapolis, IN, USA) and phosphatase inhibitor cocktail 2 (Sigma). Cellular lysates (1 mg) were immunoprecipitated, using the anti-CDC20 (5 μl, AR12, Millipore, Burlington, MA, USA) or anti-MAD2 (5 μg, A300, Bethyl Lab, Montgomery, TX, USA) antibodies. The immunoprecipitated products or total lysates were resolved on SDS polyacrylamide gel electrophoresis and transferred to PVDF membranes (BioRad). Proteins were detected with specific antibodies. The following antibodies were used in western blot analyses: anti-Cyclin B1 (H433), anti- CDC2 [54 (link)], anti-β-Tubulin (H-235, Santa Cruz Biotechnology, Santa Cruz, CA, USA); ant-MAD2 [48 (link)], anti-phosphoserine/threonine (22A, BD Biosciences); anti-β-Actin (M2, Sigma); anti-cyclin A2 (BF683), anti-cleaved Caspase 3 (D175), anti-p-RPS6 (S235/236) (2211), anti-p-CDC2 (Y15), anti-RB (4H1), anti-p-RB (S780) (D59B7), anti-APC2 (12301, Cell Signaling Technology, Danvers, MA, USA); anti-MPM2 (Millipore); anti-CDC20 (A301, Bethyl Lab). Horseradish peroxidase-conjugated anti-mouse or anti-rabbit IgG antibodies (Cell Signaling Technology; GeneTex, Irvine, CA, USA) were used and proteins were visualized with an enhanced chemiluminescence system (Advansta, Menlo Park, CA, USA).

Cells were lysed in RIPA buffer (150 mM NaCl, 0.5% sodium deoxycholate, 1% NP40, 0.1% SDS, 50 mM Tris-HCl (pH 8.0)) containing a protease inhibitor and a phosphatase inhibitor cocktail (Roche, Basel, Switzerland). A cell extract of 100 μg was separated using 10% SDS-PAGE and transferred to PVDF membranes, which were incubated with anti-PeV-A VP0 polyclonal antibody (VP0 pAb, 1:500), mouse antiserum, or hybridoma cell supernatant, then HRP-conjugated goat anti-rabbit or mouse IgG secondary antibody (Jackson ImmunoResearch Laboratory, West Grove, PA, USA), and visualized using an enhanced chemiluminescence system (Advansta, San Jose, CA, USA). Images were acquired using a digital image system (UVP, LLC).