Fig. 3.

Spatiotemporal characteristics of GnRH and PDBu-stimulated ERK regulation revealed using an ERK knock-down and add-back model. Cells were transfected in 96-well plates with ERK1/2 siRNAs and transduced with Ad ERK2-GFP and Ad mGnRHR prior to stimulation with 10⁻⁶ M GnRH or PDBu for the times indicated. They were then fixed and stained before image acquisition and analysis for the calculation of whole-cell ppERK2 intensity (upper left panel) and the N:C ERK2-GFP ratio (lower left panel). Representative regions of cell images are also shown for DAPI, ERK2-GFP and ppERK2 in cells stimulated with 10⁻⁶ M GnRH or PDBu as indicated (right panels). Note that in spite of comparable initial responses appreciable levels of ppERK2 and nuclear retention of ERK2-GFP are only seen at 120 min in the PDBu stimulated cells (scale bars: 30 μm).

Adapted from Armstrong et al. (2009c).