Immunoblotting of Protein Lysates

Cell lines were lysed in RIPA buffer supplemented with cOmplete protease inhibitors (Roche), PhosSTOP phosphatase inhibitors (Roche), and 0.1% benzonase (Novagen) on ice for 60 minutes. Lysates were clarified by centrifugation at 20,000 x g for 10 minutes at 4 °C and immunoblotting was performed using an Odyssey CLx Imager (LI-COR) as previously described.^{26 (link)} The following primary antibodies were employed in this study: HA (Cell Signaling, #3724 and #2367), DCLK1 (Abcam, #ab31704), phospho-ERK1/2 T202/Y204 (Cell Signaling, #4370), ERK1/2 (Cell Signaling, #4696), phospho-AKT S473 (Cell Signaling, #4060), AKT (Cell Signaling, #2920), FKBP12 (Abcam, #ab24373), KRAS^G12V (Cell Signaling, #14412) and α-Tubulin (Cell Signaling, #3873). Fluorescently labelled infrared secondary antibodies (Licor, IRDye) were employed as appropriate.