Fig. 4. Rolipram leads to reduced RhoA 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. The level of active GTP-RhoA was determined by incubating lysates with GST-C21 and analysing the amount of bound RhoA on a western blot. Aliquots of total lysates were also probed with anti-RhoA antibodies to control for total amount of RhoA protein. The level of active RhoA was quantified by densitometric analysis of western blots – the amount of active RhoA was normalised to the amount of total RhoA. Results shown are from cells plated in the absence (black bars) or presence (white bars) of rolipram and of three independent experiments±s.e.m. (B) Cells were left untransfected, transfected with a dominant negative RhoA construct (N19RhoA) or treated with exoenzyme C3 transferase (10 µg/ml in media for 24 hours). Cells were then plated onto laminin under control conditions, fixed 1 hour after plating and stained for actin using TRITC-phalloidin. Arrows indicate actin microspikes. N19RhoA transfectants were identified using an antibody that recognises the N-terminal Myc tag of this protein (not shown). Scale bar, 20 µm. (C) Cells were allowed to adhere to laminin coated chamber slides in the presence of carrier control DMSO (0.1%), LPA (20 µg/ml), rolipram (10 µM) or LPA plus rolipram. Cells were fixed 1 hour after plating and stained for Scar1 (green) and actin (red). Scale bar, 40 µm. The lower panel represents a quantification of cells displaying protrusive microspikes under each condition. Results represent the mean±s.e.m. of three independent experiments.