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Figure 2 | Proteome Science

Figure 2

From: Spatial proteomic and phospho-proteomic organization in three prototypical cell migration modes

Figure 2

Further characterization of pseudopod purification system. (A,B) Cell body and pseudopod morphologies in the different cell migration systems. Cells were allowed to attach for 1 hour, then treated with or not with chemoattractants for another hour. Filters were then fixed and stained. Pseudopods (bottom filter) or cell bodies (top filter) were then scratched away; filters were mounted and evaluated using epifluorescence microscopy with a 60x high numerical aperture. Representative micrographs indicating nuclei in blue (DAPI), tubulin in red (α-tubulin), and F-actin in green (phalloidin) are shown. Single channels in inverted black and white contrast, as well as color composites are shown. Bar = 20 µm. (A) Cell body morphology. (B) Pseudopod morphology. (C,D) Quantification of relative cell body (top filter surface) or pseudopod (bottom filter surface) spreading. Region of interest in the images shown in (A,B) were manually drawn, and area was measured. Area was then normalized to the average cell body or pseudopod areas observed in haptokinesis. Average as well as standard deviations are shown. Statistical significance is indicated (T-test, **: p < 0.05, n.s.: non-significant). (C) Cell body area. (D) Pseudopod area. (E,F) Biochemical analysis of pseudopod and cell body fractions in the different cell migration modes. Equal amounts of cell body and pseudopod fractions were analyzed. (E) Coomassie stained gel of pseudopod and cell body fractions in the different cell migration modes. (F) ERK activation status in the different pseudopod and cell body fractions. Different fractions were probed with anti phospho-ERK and total-ERK antibodies. Note that phospho-ERK and total-ERK signals comes from two independent western blots. Raw data is shown in supplementary Figure S5.

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