Fig. 4

Applications of SH2-PLA assay. a, Practical limit of detection from cell culture. Two-fold serial dilutions of A431 cells were seeded to wells in a 96-well plate. Image series shows various 10x magnifications of diluted A431 cells with cell number indicated. Vav2 SH2:pEGFR interaction of starved or EGF-stimulated cell lysates was quantified by the SH2-PLA assay to resolve the assay detection limit. The Ct values from real-time PCR are shown with approximate numbers of cells per culture well or per assay (in brackets) underneath the chart. b , Time course and dose response of EGF stimulation. A431 and Cos1 cells were starved and stimulated with EGF at various times and concentrations as indicated. Upper panel shows far-Western blotting with Grb2 SH2 (25 μg lysate loaded per lane) and control blotting with anti-actin. Bottom panel shows Ct values of comparable SH2-PLA experiments loading 0.4 μg lysate per assay well. c , Correlation between far-Western and SH2-PLA assay. Using experimental results shown in B, EGFR band intensities of far-Western blot (X-axis) and average Ct values of SH2-PLA (Y-axis) in panel B were plotted and showed strong correlation. a.u., arbitrary unit; r, Pearson correlation coefficient. d , Application of SH2-PLA for cancer tissue analysis. The SH2-PLA/Western/far-Western analyses were performed using 10 lung cancer tissue samples. Upper panel shows Western and far-Western results with antibody/probe names indicated on the left. Only one sample (#3) shows an EGFR size band which also overlapped with bands detected by anti-pTyr and Grb2 SH2 (far-Western image represents 60-min exposure). The tyrosine phosphorylation level of the band is similar to the weak phosphorylation of EGFR in unstimulated A431 cells (right panel). The PLA-SH2 results for the same set of lung cancer samples are shown on the bottom. Consistent with the Grb2 far-Western result, only sample #3 had significant signal beyond the no protein control (NPC). The BG line indicates the background Ct value