Cell lysates were made as described above. Lysates were incubated with various concentrations of biotinylated scrambled-palmitoylated, unpalmitoylated C1025 containing EGFR C-terminal tail, and palmitoylated C1025 containing EGFR C-terminal tail peptides dissolved in DMSO overnight at 4°C. Palmitoylated peptides were synthesized with diaminopimelic acid (DAP) substituted for cysteine, which was conjugated to palmitate by an ether linkage, making the modification resistant to thioesterase cleavage (Biomatik, USA). The streptavidin agarose beads (Thermo Scientific) were washed three times with aforementioned lysis buffer. Lysates with peptides were incubated with 20 μl of the prewashed streptavidin agarose beads for 2 hours at 4°C with rotation. The beads-lysate-peptide mix was spun down at 6000g for 1 min. The beads were washed three times with cold lysis buffer. Loading sample buffer containing β-mercaptoethanol was added to beads, and the beads were then boiled for 10 min at 100°C. The boiled sample was centrifuged at 16,000g for 1 min, and the supernatant was collected for Western blotting.

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