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Fig. 3. The ARAP3-knockdown phenotype can be rescued and mimicked. (A) 52B3 cells were seeded onto glass coverslips, starved for 16 hours and then microinjected with the injection marker biotin-dextran and pCMV3(EE) (dextran) or with biotin-dextran and pCMV3(EE)hARAP3 (hARAP3). Where indicated, cells were stimulated with PDGF for 5 minutes prior to fixation. Cells were stained for the presence of the injection marker (not shown), for F-actin (left) or for paxillin (right). The rescue result shown was observed in three independent experiments in 35% of ARAP3-injected cells. 45% of hARAP3-injected cells displayed the ARAP3-overexpression phenotype with retractions and loss of adhesion that has been previously described (Krugmann et al., 2004). (B) PAE cells were transiently transfected with pcDNA3-HA-L67Arf6 and/or pRK5-Myc-L63RhoA and the empty vector where applicable, and seeded onto glass coverslips. Cells were serum-starved for 16 hours prior to stimulation with PDGF for 5 minutes or its vehicle (indicated PDGF and starved). Cells were fixed and stained for the HA and Myc epitope tags (not shown; in cells co-transfected with Arf6 and RhoA constructs, in excess of 90% of cells expressed both plasmids if they expressed L67Arf6) or for the HA or Myc epitope tag together with filamentous actin. Arrows indicate Arf6-typical, actin-rich protrusions. The shown cells are representative examples; experiments were repeated three times. Bars, 20 µm.
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