Fig. 3
Phospho kinase arrays were used on the transduced BaF3 cells to validate the canonical JAK/STAT and MAPK/ERK signaling pathways downstream of the activated and non-activated receptors all as described in the Methods section. (a) BaF3 cells expressing normal and mutated G-CSFRs were serum starved for 6 hours and induced with 40 ng/mL G-CSF for 12.5 min and 90 min before the cells were lysed and the array membranes were treated with the lysates. The lysates treated membranes were exposed to primary and secondary antibody cocktail provided by the vendor and signal was measured with ChemiDocTM touch imaging system for phospho-Stat5, phospho-Stat3, and phospho-Erk1/2 (panels: a–c respectively). The full images of the membrane blots are also provided as Supplementary Fig. 1. The quantitative data analysis was performed using Progenesis SameSpots software with the relative spot volumes plotted in the right panels for the indicated phosphorylation sites.
