Extended Data Fig. 3: Orthogonal validation of p-ERK/c-Myc interaction and expression in HCC827 cells.
a, Schematic illustration of measuring p-ERK/c-Myc interaction using co-IP and PLA, Created with BioRender.com. b, Top Panel of western blots depicted results of co-IP of p-ERK and c-Myc in c-Myc pull-down samples. The cells were treated with 100 nM Osimertinib for 0, 8 12 hours. N-IgG served as a negative control for co-IP. The bottom panel demonstrated the results of p-ERK, c-Myc, and GAPDH expression run on different gels from the same HCC827 cell lysate. GAPDH was used as a negative control. P-ERK expressions at short and long exposure were shown in the gel. c, Workflow of measuring 9 phosphorylated proteins in HCC827 cells using Luminex. Created with BioRender.com. d, Quantification of 9 phosphorylated proteins in HCC827 cells treated with and without 12-hour 100 nM Osimertinib was shown in the heatmap and bar graph. Bar graphs are shown as mean ± 1 SD. e, Quantification of p-ERK/c-Myc PPI counts in different ROIs with a high, low, and medium expression of p-ERK/c-Myc in HCC827 cells. The right graph shows the cumulative density which measures the percentage of cells expressing different numbers of p-ERK/c-Myc events per cell across three ROIs. The detailed statistics for each ROI are shown in Supplementary Fig. 25. Box plots: median (horizontal line inside box), 25th and 75th percentiles (box), 25th and 75th percentiles ±1.5 times the interquartile range (whiskers).
