Anti mouse igg horseradish peroxidase hrp

Surface biotinylation and immunoblotting was performed as described previously. 22 Transfected HEK293T cells were washed three times with ice-cold phosphate-buffered saline (PBS). The cells were then treated with sulfo-NHS-SS-biotincontaining buffer (Pierce, Rockford, IL, USA) for 30 minutes to biotinylate the plasma membrane proteins. After biotinylation, the cells were washed with quenching buffer to remove the excess biotin and washed twice again with PBS. The cells were harvested and incubated overnight with avidin solution (Ultra-Link Immobilized NeutrAvidin Beads 10%, Pierce) at 48C. Avidin-bound complexes were washed three times and the biotinylated proteins were eluted in a 23 sample buffer. The protein samples were suspended in a sodium dodecyl sulfate buffer and separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The separated proteins were transferred to a nitrocellulose membrane and blotted with appropriate primary and secondary antibodies. Anti-Myc (Santa Cruz Biotechnology, Santa Cruz, CA, USA) antibody was used as primary antibody, and anti-Mouse IgG Horseradish peroxidase (HRP) (Thermo Scientific, Rockford, IL, USA) antibody was used as secondary antibody. Protein bands were detected by enhanced chemiluminescence (Amersham Biosciences, Buckinghamshire, UK).