For immunoblot analysis of FGFR1 and the α-subunit of the NaK-ATPase, cells were collected in phosphate-buffered saline, centrifuged (3min at 3000 rpm) and suspended in lysis buffer. Aliquots of the cell lysates containing 150 μg of protein were submitted to SDS-PAGE and immunoblotted for FGFR1 (Origene, Rockville, MD) and NaK-ATPase α-subunit (sc-21712) (Santa Cruz Biotechnology, Santa Cruz, CA). Densitometry was performed using Quantity One® for FGFR1 and NaK-ATPase immunoblots where NaK-ATPase was measured as a loading control.
For immunoblot analysis of phospho-ERK and total ERK, cells were plated at 25,000 cells/well in a 6-well plate. 24 hours later, cells were switched to HITES media for 2 hours and subsequently treated with concentrations of ponatinib (0–1μM) for 2 hours. Cells were rinsed in phosphate-buffered saline, lysed in 250 μl lysis buffer and aliquots were submitted to SDS-PAGE and immunoblotted for phospho- ERK (P-p44/42 MAPK) (Cell Signaling Technology, Danvers, MA) and ERK 1 (sc-93) and ERK 2 (sc-154) (Santa Cruz Biotechnology, Santa Cruz, CA).
For immunoblot analysis of phospho-ERK and total ERK, cells were plated at 25,000 cells/well in a 6-well plate. 24 hours later, cells were switched to HITES media for 2 hours and subsequently treated with concentrations of ponatinib (0–1μM) for 2 hours. Cells were rinsed in phosphate-buffered saline, lysed in 250 μl lysis buffer and aliquots were submitted to SDS-PAGE and immunoblotted for phospho- ERK (P-p44/42 MAPK) (Cell Signaling Technology, Danvers, MA) and ERK 1 (sc-93) and ERK 2 (sc-154) (Santa Cruz Biotechnology, Santa Cruz, CA).