Erk1 k23

HEK 293 cells were cultivated to 70–80% confluency before transfection. For transfection of Fgf8 and Hsc70 cells were seeded at 2×10⁶ cells in a 10cm plate. After 24 hours cells were transfected according to the manufacturer’s protocol using Promofectin (Promocell). Briefly two mixtures containing DNA/serum free DMEM and Promofectin/serum-free DMEM were combined incubated for 20 min at RT and added to the cells. 24 hrs after transfection the culture medium was replaced and Bafilomycin A1 was added. 24 hrs later the cells were lysed in sodium dodecyl sulfate (SDS)–sample buffer containing 100 mM dithiothreitol (DTT) and subjected to Western blot analysis. Activated Erk was monitored using an antibody against phosphorylated Erk (phospho-p44/42, Cell Signaling). For the loading control the membrane was stripped (62.5 mM Tris, pH 6.8, 2% SDS, 0.8% DTT) and reprobed with an Erk antibody (Erk 1 (K-23) Santa Cruz). Blots were stained using the enhanced chemiluminescence system (Thermo Fisher Scientific). The quantification of proteins bands in Western blot analysis was performed using the program ImageJ.