Figure 2

CEER's single cell sensitivity and capacity for functional profiling of pathway proteins. (a) The activation of HER1 and HER2 at a sensitivity level of a single cell in MDA-MB468 and SKBr3 respectively are shown above. These cell lines express approximately 1 to 2 × 10⁶ HER1 or HER2 RTKs on their cell membrane per cell. Microarray slide images for 3 cells, 1 cell, 0.3 cell and negative control are shown above the cell titration curve. MDA-MB468 cells were treated with EGF to activate HER1 while HER2 is constitutively phosphorylated in SKBr3 cells. The cell amount on each pad was generated by serial dilution. Capture antibodies were printed with 500 pl per spot in triplicates in serial dilutions of 1.0 mg/ml, 0.5 mg/ml, 0.25 mg/ml and 0.125 mg/ml. Relative signal intensity scale is shown for reference. (b) The western blot data were generated from 12 μg of total protein per lane. The level of dominant RTK expression in each cell line was determined pre- and post- EGF or HRG stimulation. The CEER determined per-cell RTK activation (RFU/cell) level for each cell line is summarized. Non-detectable signals in each cell lines were indicated as ND in the table. (c) Detection of pHER1, pHER2, total HER1 and total HER2 in SKBR3 and MDA-MB-468 cells using CEER or ELISA assays are shown. Sensitivity of CEER and ELISA assays were shown by detection limit, which was defined as cell numbers when signal noise ratio (s/n) > 3. pHER1 in MDA-MB-468 cells (top left), pHER2 in SKBR3 cells (top right), HER1 in MDA-MB-468 cells (bottom left) and HER2 in SKBR3 cells (bottom right). Closed square (red): HER2s or HER1s (RFU) detected by CEER assay; Closed circle (blue): HER2s or HER1s (OD450) detected by ELISA assay.