Figure 3. Signal linearity with the in-cell western assay.

A. 96-well microplates were loaded with an increasing number of cells/well. Signals from anti-GAPDH and anti-PMLC₂₀ antibodies appear as green fluorophores. Signals from cell dyes (cell number normalization) appear as red fluorophores. B. Quantification of signals shown in panel A. Normalized values are expressed as a fraction of the signal intensity of the wells containing 15,000 cells for GAPDH (•), PMLC₂₀ (▴). The signal from the cell dyes also is shown (▪). The GAPDH and PMLC₂₀ signals appear to plateau at the higher cell densities so the line of best fit has been calculated and illustrated by expressing the normalized values as a fraction of the intensity of the wells containing 10,500 cells (inset) for GAPDH (•), and PMLC₂₀ (▴).