Wet western blot transfer

Protein samples were prepared from cells using standard methods and were subjected to SDS-PAGE and transferred to a nitrocellulose membrane by wet western blot transfer (Bio-Rad) [12 (link)]. The membrane was blocked in TBST buffer (TBS + 1% Tween) containing 5% milk for 1 hour at room temperature. The blocked membranes were incubated overnight at 4°C with specific primary antibodies (Odyssey blocking buffer, 927–40000). The membranes were washed 3 times with TBST buffer, and then incubated with specific secondary antibodies provided by LI-COR for 2 hours at room temperature. After 3 washes with TBST buffer, the membranes were analyzed using the ODYSSEY Infrared Imaging system (LI-COR). Analysis of biotinylated cell surface proteins was performed using the Pierce Cell Surface Protein Isolation Kit (89881) from Thermo Scientific (Waltham, MA) [12 (link)]. Briefly, cells were labeled with Sulfo-NHS-SS-Biotin, a thiol-cleavable amine-reactive biotinylation reagent, and then lysed with mild detergent. Biotinylated surface proteins were isolated with Avidin Agarose, and eluted using SDS-PAGE sample buffer containing 50mM DTT. Samples were then analyzed for HER2 by immunoblot as above. All immunoblot experiments were performed at least 3 times and representative blots are shown in the figures.