Cyclin a h 432 sc 751

Transfected cells were collected posttransfection and washed twice in Dulbecco's phosphate buffered saline (dPBS). Cells were lysed directly in 25 μl dPBS and 25 μl sample buffer (120 mM Tris-HCl [pH 6.8], 5% SDS, 10% 2-mercaptoethanol, 20% glycerol, 0.01% bromophenol blue), boiled for 10 minutes and separated by SDS-PAGE electrophoresis. Proteins were transferred to nitrocellulose membranes (Amersham Hybond-C Extra; GE Healthcare) and detected with the corresponding primary and secondary antibodies. The following primary antibodies were used; cyclin A (H-432) (sc-751; Santa Cruz), actin (I-19) (sc-1616; Santa Cruz), GFP (ab6556; Abcam), MNV-1 anti-NS1-2 [13 ] and MNV-1 anti-NS5 [13 ]. Secondary antibodies used were 680RD donkey anti-goat IgG (926–68074; LI-COR) and 800CW donkey anti-rabbit IgG (926–32213; LI-COR).

Lysates were prepared from pellets using RIPA buffer and sonication, following standard protocols. Western blots were run on precast Any KD gels (Bio-Rad Labororatories Co., Ltd., Hercules, CA, USA) in accordance with standard protocols and transferred using the Bio-Rad Turbo blotter high molecular weight program. K-Ras (F234, sc-30, 1:500), N-Ras (F155, sc-31, 1:1000), Cyclin A (H-432, sc-751, 1:1000), B (H-433, sc-752, 1:1000), and E (C-19, sc198, 1:1000) were all purchased from Santa Cruz Biotechnology (Heidelberg, Germany) and Cyclin D (ab137875, 1:1000) was purchased from AbCam (Cambridge, UK). RNA-induced silencing complex (RISC) free control was from GE healthcare/Dharmacon Lafayette, CO, USA (D-001600-01-05). The membranes were blocked in 5% milk Phosphate Buffered Saline with 0.1% Tween (PBST 0.1%) for three hours before being incubated with primary antibody in 5% milk, PBST 0.1% at 4 °C overnight. Six ten-minute washes in PBST 0.1% were followed by incubation of the membrane with an HRP conjugated secondary anti-mouse antibody (sc-2357). Following incubation with a secondary antibody, six ten-minute washes with PBST 0.1% were carried out, and the membrane was visualised using chemo-luminescence and developed (Western lightning plus ECL Life Technologies, Paisley #31985-062).