Reverse transfection of cells was performed with jetPEI. Plasmid DNA (5 μg, 50 μl) was mixed with NaCl (500 μl, 150 mM). JetPEI (15 μl) was separately mixed with the same amount of NaCl (150 mM). The jetPEI mixture was then added to the DNA mixture and incubated at room temperature for 15 min. Cells (1 million) were trypsinized and suspended in complete cell culture medium. Finally, the prepared transfection complex mixture was added to the cells and seeded onto tissue culture dishes.

Understanding receptor activation is important for disease intervention. Activation of the receptor tyrosine kinase c-KIT is involved in numerous diseases including melanoma, mastocytosis, multiple myeloma and gastrointestinal stromal tumors. To better understand the regulation of activation, we studied the two c-KIT isoforms, c-KIT(-) and c-KIT(+), which differ by a tetrapeptide insert GNNK, located in the extracellular juxtamembrane domain of the c-KIT(+) isoform. This region is important for regulating receptor activation. Here we show that the consecutive elimination of one amino acid at a time from the GNNK tetrapeptide insert gradually increases receptor tyrosine phosphorylation, ubiquitination, internalization and downstream MAP kinase-ERK activation. Successively decreasing the insert length progressively improves cell survival during drug treatment. Our results indicate that the length of the tetrapeptide fine-tunes receptor activity, thus providing deeper insight into c-KIT activation.