Hrp conjugated anti mouse or anti rabbit igg antibody

Whole-cell lysates of MDCK cells were electrophoresed through sodium dodecylsulfate polyacrylamide gels (Bio-Rad Laboratories, Hercules, CA) and transferred to a PVDF membrane (Millipore, Billerica, MA). The membrane was then blocked with Blocking One (Nacalai Tesque, Kyoto, Japan) and incubated with polyclonal rabbit anti-GFP (MBL, Nagoya, Japan), mouse anti-NS1 (188/5, a gift from Prof. H. Kida, Hokkaido Univ., Sapporo, Japan), rabbit anti-WSN (R309, prepared in our laboratory) or mouse anti-β-actin (A2228; Sigma-Aldrich), followed by HRP-conjugated anti-mouse or anti-rabbit IgG antibody (GE Healthcare, Waukesha, WI), respectively. After the membrane was washed with PBS-Tween, specific proteins were detected by using the ECL Plus Western Blotting Detection System (GE Healthcare). The specific protein bands were visualized by use of the VersaDoc Imaging System (Bio-Rad Laboratories).

The 293T cells that were transfected with a plasmid expressing FLAG-tagged NP (or co-transfected with the indicated NP construct and HA-tagged hnRNP A2 expression plasmid) were washed with PBS and then lysed in a lysis buffer containing 50 mM Tris-HCL (pH 7.4), 150 mM NaCl, 1 mM EDTA and 1% Triton X-100. After centrifugation at 12,000 X g for 30 minutes, the supernatants were collected and mixed with anti-FLAG M2 affinity gel (Sigma-Aldrich). The mixture was rotated at 4°C for 2 h, and the affinity gel was then collected after centrifugation at 8,000 X g for 30 seconds. The affinity gel was thrice washed with a buffer containing 50 mM Tris-HCl (pH 7.4) and 150 mM NaCl. The FLAG-tagged NP and the associated proteins on the affinity gel were eluted with 3X FLAG peptide (Sigma-Aldrich) in wash buffer. The eluted proteins were then separated on SDS-polyacrylamide gels and transferred to PVDF membranes. After blocking with 5% skim milk, the membranes were reacted with anti-FLAG (1:1000 dilution, Sigma-Aldrich), anti-HA (1:1000 dilution, Sigma-Aldrich), anti-hnRNP A1 (1:1000 dilution, Santa Cruz Biotechnology), anti-hnRNP A2/B1 (1:1000 dilution, Sigma-Aldrich) or anti-β-actin antibodies (1:2000 dilution, EMD Millipore) and then detected with an HRP-conjugated anti-mouse or anti-rabbit IgG antibody (1:5000 dilution, GE Healthcare Life Sciences).