Goat anti rabbit irdye 680cw

All the chemicals were purchased from Sigma-Aldrich or Tokyo Chemical Industry unless otherwise stated. 2,6-Dibromo-4H-cyclopenta[2,1-b:3,4-b′]dithiophene and 2,6-bis(trimethyltin)-4,8-didodecylbenzo[1,2-b;4,5-b′]dithiophene were purchased from Luminescence Technology Corp (Lumtec). 4,9-Dibromo-6,7(4-hexylphenyl)-[1,2,5]thiadiazolo[3,4-g]quinoxaline was purchased from Brilliant Matters. Secondary antibodies for immunoblotting, which include IRDye 800 CW goat anti-mouse (1:10,000; 925-32210) and IRDye 680 CW goat anti-rabbit (1:10,000; 925-68071) were purchased from LI-COR Biosciences. Anti-HGF (1:500; ab83760), anti-MTA2 (1:100; ab8106), anti-VCAM-1 (1:250; ab134047) antibodies were purchased from Abcam. Secondary antibody Alexa Fluor 488 conjugated goat anti-rabbit IgG H&L (1:500; ab150077) for immunofluorescent staining was purchased from Abcam.

The protein concentration was determined by the Bradford method against known standards. The expression and purity of protein of interest were ascertained by resolving the protein on a 12% sodium dodecyl sulphate polyacrylamide gel (SDS–PAGE), followed by Coomassie G250 blue silver staining. The specificity and integrity of proteins were determined by an immunoblotting assay using mouse antibodies specifically against CPB2 toxin (self-generated by Abmart as mentioned in Section 2.5), followed by a secondary IRDye 800CW goat anti-mouse or IRDye 680CW goat anti-rabbit (LI-COR). Immune complexes were detected using the Odyssey dual-infrared fluorescence system. Antibodies against human beta-actin, caspase-3, and Bax were products of proteintech (Rosemont, IL, USA).

For Cdc20 and BubR1 immunoprecipitation, 2 μg of plasmid was transfected into HeLa cells in a 15 cm dish 48 h before collection. Cells were synchronized by double thymidine arrest followed by overnight treatment by nocodazole (200 ng/ml). Mitotic cells were shaken off the plate and lysed on ice in a lysis buffer containing 10 mM Tris HCl, pH 7.4, 150 mM NaCl, 0.5 mM EDTA, and 0.5% NP40 with protease and phosphatase inhibitors (Roche). The cell lysate was centrifuged at 17,000 g for 10 min at 4 °C, and the supernatant was applied to 20 μl of GFP-Trap beads (Chromotek) and shaken for 2 h at 4 °C. The beads were washed three times with 0.5 ml lysis buffer and boiled in 50 μl 2× SDS loading buffer. For Cdc20 knocking out examination, cells from a 10 cm dish were collected and lysed in 200 μl of lysis buffer as described above. The cell lysate was cleaned by centrifugation and boiled in an SDS loading buffer. A quantitative western blot (Odyssey DLx, LI-COR) was performed to examine Cdc20 and interested proteins. Antibodies used include Cdc20 (Santa Cruz, sc-13162; Bethyl, A301-180A), BubR1 (homemade in JN lab), Mad1 (Sigma, M8069), Mad2 (homemade in JN lab), Apc7 (Santa Cruz, sc-365649), Apc15 (Santa Cruz, sc-398488) and GAPDH (Proteintech, 60004-1). Fluorophore-labeled secondary antibodies include goat anti-mouse IRDye 800CW and goat anti-rabbit IRDye 680CW (LI-COR).

Cell lysis was obtained from siRNA-transfected cells after 72 h using a mixture of RIPA buffer (Sigma, St. Louis, MO, USA) and protease/phosphatase cocktail inhibitor (Roche, Indianapolis, IN, USA). Protein concentration was measured with a BCA assay (Pierce, Bradenton, FL, USA). Equal amounts of protein (5 μg) per sample were separated via SDS-PAGE and transferred to an Immobilon-P PVDF membrane (Millipore, Burlington, MA, USA). Membranes were blocked with 5% skimmed milk. The membranes were incubated with primary antibodies, monoclonal mouse anti-human β-tubulin (Sigma; 1:1000), monoclonal rabbit anti-human ROCK1 (Cell Signaling, Danvers, MA, USA; 1:1000), and monoclonal rabbit anti-human ROCK2 (Cell Signaling; 1:1000), followed by incubation with secondary antibodies, goat anti-mouse IRDye 800CW and goat anti-rabbit IRDye 680CW (1:5000, LiCOR, Lincoln, NE, USA), at room temperature for 3 h using iBind Western Device (ThermoFisher Scientific). Next, the membranes were imaged using an Odyssey infrared imaging system (LI-COR Biosciences, Lincoln, NE, USA). A densitometry analysis of protein expression was performed using the Image J gel analysis tool.

Cell pellets from transfection procedures were lysed in Mammalian Protein Extract Reagent (M-PER; Pierce/Thermo Scientific, Rockford, IL, USA). Following pre-cleaning by centrifugation, protein concentrations of cell lysates were determined by using Protein Assay Reagent (Bio-Rad, Hercules, CA, USA). Lysates equivalent to 25 μg of protein were separated on NuPAGE Bis-Tris (4-12%) gels (Invitrogen, Carlsbad, CA, USA) and transferred onto PVDF membranes. Membranes were blocked in Blocking Buffer (LI-COR, Lincoln, NE, USA) and incubated with specific antibodies against ERG splice variants (ERG MAb 9FY; Biocare Medical Inc., Concord, CA, USA), C-MYC (Epitomics, Burlingame, CA, USA) and GAPDH (Santa Cruz biotechnology, Santa Cruz, CA, USA). Membranes were washed in Tris-Buffered Saline + Tween 20 (TBST) before incubation with appropriate secondary antibodies (goat anti- Mouse IRDye 8000CW or goat anti-Rabbit IRDye 680CW, LI-COR). Signals of proteins detected were visualized and quantitatively measured using the Odyssey infra-red imaging scanner and software (LI-COR).

Low-passage HEK293T cells, maintained in Dulbecco's modified Eagle medium (DMEM)+10% BGS+1% penicillin/streptomycin, were transfected with either control plasmid or human pCS2P-CCDC103-myc (gift from Iain A. Drummond, MDI Biological Laboratory, Bar Harbor, ME, USA) for 24 h. Samples were lysed in RIPA buffer (10 mM Tris-HCl, pH 8.0, 1 mM EDTA, 1% Triton X-100, 0.1% sodium deoxycholate, 0.1% SDS, 140 mM NaCl) and incubated at 95°C for 5 min. A volume of sample corresponding to 30 μg of total protein was separated by electrophoresis on a 4-20% gradient Tris-glycine gel (Bio-Rad) and transferred to nitrocellulose membrane, blocked in 5% BSA in Tris-buffered saline with 0.1% Tween (TBST) and incubated overnight at 4°C with 1:500 rabbit anti-CCDC103 antibody (YenZym Antibodies) and 1:2000 mouse anti-tubulin antibody (Sigma-Aldrich, T6199). Membranes were washed and incubated for 1 h in 1:15,000 goat anti-rabbit IRDye 680CW (LICOR) and 1:15,000 goat anti-mouse IRDye 800Rd (LICOR), and bands were visualized on the LICOR Odyssey imaging system.