Gapdh rabbit polyclonal

Heparan sulfate (HS) positive CHO-K1 and HS negative CHO-745 cells were infected for 3 h at 37°C with HSV-1 gL86 virus at 10 PFU/cell in presence and absence of lipofectamine (8 μg/mL). Cells were lysed in radioimmunoprecipitation buffer (RIPA, Sigma) with Proteinase and Phosphatase (Halt, Pierce Biotechnology) and electrophoresed on a 4–12% Bis-Tris Gel (NuPage). A PVDF membrane (Novex) was used for transfer. After initial non-specific blocking in 5% non-fat milk, the membrane was then incubated with primary (VP16 mouse monoclonal, Santa Cruz; GAPDH rabbit polyclonal, Santa Cruz) and secondary HRP-conjugated antibodies (Jackson). The membrane was then developed (ECL, Pierce) and visualized (ImageQuant LAS 4000, GE Healthcare).

The following antibodies were used at the indicated dilutions for western blot. Cdc20 mouse monoclonal (sc-13162, 1:1,000, Santa Cruz), Cdc20 rabbit polyclonal (A301-180A-1, 1:1,000, Bethyl Laboratories), GAPDH rabbit polyclonal (sc-25778, 1:500, Santa Cruz), APC1 rabbit polyclonal (A301-653A-1, 1:500, Bethyl Laboratories), APC3 mouse monoclonal (610454, 1:500, BD Biosciences), APC7 rabbit polyclonal (A302-551A-1, 1:500, Bethyl Laboratories), Cdc20 T70 and T59 phosphorylation specific antibodies (raised against CSKVQT(Tp)PSKPG and CRTPGR(Tp)PGKSS, respectively; 1:500 and 1:250, Moravian Biotechnology). APC3 S447p (raised against CGKISTI(Tp)PQIQAF; 1:500, Moravian Biotechnology), BubR1 S670p (raised against TLSIKKL(Sp)PIIED,1:500, 21st Century), cyclin B1 S126p (Gift from Jonathon Pines, 1:500), Anti-phospho Histone H3 (Ser 10) rabbit polyclonal (06-570, 1:1,000, Millipore), cyclin B1 mouse monoclonal (554177, 1:1,000, BD Pharmingen), cyclin A rabbit polyclonal (sc-751, 1:1,000, Santa Cruz), Vinculin mouse monoclonal (V9131, 1:10,000, Sigma), Securin mouse monoclonal (K0090-3, 1:500, MBL), PP1 gamma rabbit polyclonal (A300-906A, Bethyl, 1:500), PP2A catalytic subunit mouse monoclonal (05-421, Millipore, 1:2,500). BubR1 mouse monoclonal (raised against TPR domain, 1:500, Biotech Research and Innovation Center, Copenhagen).