Pulsed-SILAC analysis of protein translation rate changes upon protrusion formation

To assess the impact of protrusion formation on protein synthesis rates on a global scale, we carried out a pulsed-SILAC (pSILAC) analysis in MDA-MB231 cells grown with or without protrusions for 1, 2, 4, and 8 hrs. Briefly, light SILAC labelled cells were seeded on top of 2x 3 micron transwell filters without any media added to the bottom chamber. Cells were allowed to attach to the filter overnight, and the next day the media on the top was changed to medium (M) or heavy(H) SILAC DMEM. At the same time, the H SILAC media was also added to the bottom chamber of the transwell with H media on the top to induce protrusions. Cells were then allowed to form protrusions for 1, 2, 4, or 8 hrs, before being lysed and mixed with the M labelled lysates (cells without protrusions) from the same timepoint. The experiment was then repeated with switched SILAC labelling.