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Fig. 1. ARAP3-deficient cells do not form protrusive lamellipodia. (A) Western blots of lysates of stable PAE cell lines expressing a scrambled control RNAi oligonuleotide (clone NA1) or olignucleotides directed against different parts of porcine ARAP3 (clones 52B3, 32D6) were probed for ARAP3 (top panel) and subsequently reprobed for ß-COP as a loading control (bottom panel). Careful analysis of blots from several similar experiments leads us to estimate that the reduction in ARAP3 expression in the 52B3 and 32D6 lines is approximately 85%. The original blot had 25 lanes, out of which the shown ones were not immediately adjacent. They were cut and pasted for ease of viewing. (B) Cells of the stable RNAi PAE cell lines were seeded onto coverslips, starved for 16 hours, stimulated for 5 minutes either with PDGF or with its vehicle (as indicated) and fixed. Cells were stained for F-actin. An arrow points to a 52B3 cell carrying a typical small actin-rich protrusion. The photographs shown are representative examples from four independent experiments. (C) NA1, 52B3 and 32D6 cells were treated as above and 120-150 cells per coverslip were counted and scored according to their phenotypes: black bars, no ruffles or lamellipodia; striped bars, protrusive lamellipodia; grey bars, uniform ruffles along entire cell periphery; unfilled bars, dorsal ring ruffles. (D) NA1 and 52B3 cells were treated as in (B) and stained for paxillin. Arrows point to typical clustered focal adhesions in 52B3 cells. Bars in (B) and (D), 20 µm.
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