RAW264.7 cells were lysed in NP40 buffer (P0013F; Beyotime) supplemented with protease and phosphatase inhibitors. After 2 hours of incubation with agarose beads, the cell lysates were incubated overnight with immunoglobulin G or anti-SCAP antibody. The immunocomplexes were washed 3 times with PBS and collected proteins from the magnetic beads. Finally, the proteins were detected by Western blotting analysis.
Western blotting was performed as described previously.21 Total proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and then transferred to a polyvinylidene difluoride membrane (IEVH85R; Millipore, Burlington, MA). Then, the membranes were blocked with 3% bovine serum albumin for 1 hour. After primary antibodies overnight incubation, the membranes were incubated with horseradish peroxidase–labeled corresponding secondary antibodies. Finally, detection was visualized by using the ECL chemical luminescent detection kit (Bio-Rad, Hercules, CA), and the bands were further analyzed using Quantity One software. Antibodies are shown inTable 3 .
Western blotting was performed as described previously.21 Total proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and then transferred to a polyvinylidene difluoride membrane (IEVH85R; Millipore, Burlington, MA). Then, the membranes were blocked with 3% bovine serum albumin for 1 hour. After primary antibodies overnight incubation, the membranes were incubated with horseradish peroxidase–labeled corresponding secondary antibodies. Finally, detection was visualized by using the ECL chemical luminescent detection kit (Bio-Rad, Hercules, CA), and the bands were further analyzed using Quantity One software. Antibodies are shown in