800cw donkey anti rabbit igg

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).

All antibodies used in this study are listed in S2 Table. Immunoblotting was performed as described [27 (link)]. Briefly, tissues were homogenized in ice-cold RIPA lysis buffer (250mM Tris-HCl, pH 7.4, 750 mM NaCl, 5% Triton X-100, 2.5% sodium deoxycholate, 0.5% sodium dodecyl sulphate (SDS), 100 mM NaF, 2 mM Na₃VO₄, 1 mM phenylmethylsulfonyl (PMSF) and 1% cocktail protein protease and phosphatase inhibitors (Sigma), pH 8.0). Lysates were centrifuged at 14,000x g for 30 min at 4°C, and supernatants were stored at -80°C until analysis. Lysate protein was resolved by SDS-PAGE (Novex NuPAGE 4–12% Bis-Tris gels, Invitrogen) and transferred to polyvinylidene difluoride (PVDF) membranes (Millipore sigma). Phosphorylated and total proteins were identified by immunoblotting using the following primary antibodies: Phospho-S6 Ribosomal Protein (Ser240/244) Antibody #2215 and α-Tubulin (DM1A) Mouse mAb #3873 (Cell Signaling Technologies) at 1:1000 dilution. Immunoblots were evaluated using IRDye® 680RD donkey anti-mouse IgG and 800CW donkey anti-rabbit IgG (LI-COR) at 1:15,000 and scanned on an Odyssey Clx Imaging System (LI-COR). Fluorescent signals were quantified using Image Studio Software (LI-COR).