Eu 88 image scanner

For Western blot analysis, cells were washed with PBS and lysed in RIPA lysis buffer (Bioss, Beijing, China). Next, total protein (200 μg) was separated by 12% SDS-polyacrylamide gel electrophoresis (SDS-PAGE), and transferred to a polyvinylidene fluoride (PVDF) membrane (Millipore Corporation, Billerica, MA, USA). The PVDF membrane was blocked with 5% nonfat milk at room temperature for 1 h, followed by incubation with the appropriate specific primary antibodies overnight at 4 °C. The PVDF membrane was then rinsed with Tris-Buffered Saline Tween-20 (TBST) and stained with the appropriate horseradish peroxidase (HRP)-labeled secondary antibody for 1 h at room temperature. After washing with TBST, proteins were visualized with Electrochemiluminescence (ECL) reagent (Amersham Pharmacia Biotech, Piscataway, NJ, USA).
For immunoprecipitation analysis, the cells were lysed with IP lysis buffer, and the total protein (5 μg) was immunoprecipitated with anti-MyoF and anti-Dvl-2 antibodies. Immunocomplexes were washed three times with IP lysis buffer and analyzed by Western blotting as described. Quantification of protein blots was performed with the Quantity One 1-D software (version 4.4.0) (Bio-Rad, Hercules, CA, USA) using images acquired from an EU-88 image scanner (GE Healthcare, King of Prussia, PA, USA).