Amersham imager 600 ccd based chemiluminescent analyzer

Cells were washed three times with cold phosphate-buffered saline (PBS) and lysed in cold lysis buffer (1% Triton X-100, 1 mM phenylmethylsulfonyl difluoride [PMSF] in PBS) for 30 min. Lysates were clarified by centrifugation at 12,000 × g for 10 min. Proteins in the lysates were separated by SDS-PAGE, transferred to nitrocellulose membranes (10600001; GE Amersham), and then probed with the antibodies indicated in the figure legends. Finally, the membranes were incubated with enhanced chemiluminescence (ECL) reagents (Vazyme, China), and the signals were analyzed using an Amersham Imager 600 CCD-based chemiluminescent analyzer (GE Healthcare). Proteins were detected with the following antibodies: anti-GAPDH (10494-1-AP; proteintech); anti-PB2, anti-PB1, anti-PA and anti-NP antibodies (kindly provided by Dr. Chengjun Li, Harbin Veterinary Research Institute, The Chinese Academy of Agricultural Sciences); anti-DDX39B (14798-1-AP; proteintech)l; anti-DDX39A (11723-1-AP; proteintech); anti-CIP29 (15798-1-AP; proteintech)l; anti-THOC1 (10920-1-AP; proteintech); anti-THOC4 (16690-1-AP; proteintech); anti-FLAG MAb (F1804; Sigma-Aldrich); rabbit anti-FLAG PcAb (9121; HUABIO); anti-MYC MAb (60003-2-Ig; proteintech); and anti-HA MAb (H9658; Sigma-Aldrich).

The NSCs treated in each experiment were homogenized in SDS buffer (4% SDS, 125 mM Tris–glycine, 10% 2-mercaptoethanol, 2% bromophenol blue in 30% glycerol) and centrifuged at 9,000 g for 10 min at 4 °C to remove debris. Aliquots were subjected to polyacrylamide gel electrophoresis in the presence of SDS (SDS/PAGE) followed by electrotransfer onto a PVDF membrane (Hybond-P; GE Healthcare, Tokyo, Japan). The blotted membranes were blocked overnight with Block Ace (UKB80, Dainippon Sumitomo Pharma) and then probed with primary antibodies overnight at 4 °C. Detection was performed with horseradish peroxidase (HRP)-conjugated secondary antibodies (all from Santa Cruz Biotechnology) and either ECL prime (GE Healthcare) or Immunostar ^® LD (290-69904, Wako) detection reagents. The lumino-labeled membranes were analyzed on an Amersham™ Imager 600 CCD-based chemiluminescent analyzer (GE Healthcare). For assessment of protein expression levels, relative band intensities were quantified using ImageQuant™ TL software (GE Healthcare). All data were reported as mean values ± SD of 3 experiments.