Fig. 5. Rolipram does not alter Cdc42 or Rac1 activity in REF52 cells. (A) REF52 cells were allowed to adhere to laminin coated plates in the absence (–) or presence (+) of rolipram. Cells were lysed 1 hour after plating and the level of active GTP-Cdc42 was determined by incubating lysates with GST-PAK and analysing the amount of bound Cdc42 by western blotting. Aliquots of total lysates were also probed with anti-Cdc42 antibodies to control for total amount of Cdc42 protein. The level of active Cdc42 was quantified by densitometric analysis of western blots – the amount of active Cdc42 was normalised to the amount of total Cdc42. Results of three independent experiments±s.e.m. are shown from cells plated in the absence (black bars) or presence (white bars) of rolipram. (B) Cells transfected with an active mutant of Cdc42 (V12Cdc42) were fixed and stained for Scar1 (blue) and actin (red). V12Cdc42 transfectants were identified with an antibody that recognises the N-terminal Myc-tag of this protein (not shown). In a parallel experiment, non-transfected cells were plated onto laminin, fixed 1 hour after plating and stained for Scar1 (blue) and actin (red). Arrowheads indicate filopodia, which differ structurally from actin microspikes (arrows). Scale bar, 20 µm. (C) Experimental set-up like in (A) except that a Rac1-specific antibody was in the western blot. (D) Cells were transfected with a dominant-negative mutant of Rac1 (N17Rac1). Cells were plated onto laminin in the presence of rolipram. Cells were fixed 1 hour after plating and stained for transfected protein (anti-Myc, green) and actin (red). Solid arrow indicates extensive lamellipodia present in untransfected cell but absent from cell expressing N17Rac1 (dotted arrow). Scale bar, 20 µm.