Figure 5
The Linearity tests for Quantifying Cellular Signaling Kinases. A series of negative (-) and positive (+) controls for phospho-specific and caspase 3 antibodies were mixed proportionally to generate artificial gradient of target proteins for the linearity tests using laser-amplified Qdot-RPPA. Examples shown here are (A) pAKT, (B) pERK, (C) pGSK3, (D) pNFkB, (E) pp38, (F) cleaved caspase 3. pAKT+, pERK+ and pGSK+ are Jurkat cells treated with phosphatase inhibitor, calyculin A, to accumulate these kinases in phosphorylated states. pNFkB+ is HeLa cells stimulated with TNF (20 ng/ml, 5 min) to activate NFkB. p-p38+ is C6 glioma cells treated with anisomycin to activate p38. Cleaved caspase 3+ is cytochrome c treated Jurkat cell lysates to induce the cleavage of caspase 3. X axis is the gradient (25% intervals) of targeted protein. Y axis is the calibrated signal intensity after subtracting the background signal at the 0% gradient. R2 values of linear regression curves close to 1 indicate laser-amplified Qdot-RPPA can distinguish at least 25% change of protein levels within the linear range. Error bars were derived from the three replicates of each sample.