Fig. 3. PKC-deficient REF cells disassemble stress fibres and focal adhesions under serum-free conditions. (A) REF cells were transfected with control siRNA oligonucleotide (–ve) or siRNA oligonucleotides 2 and 4 at the indicated concentrations. 48 hours after transfection, cells were lysed and immunoblotted for PKC and actin. The amount of PKC relative to the control, normalized to the actin content, is indicated below each lane. (B) REF cells were transfected with control (–ve) or siRNA duplex 4 (oligo 4) and serum starved for 16 hours. Cells were subsequently left untreated (–) or treated with LPA for 30 minutes (+LPA), fixed and stained for F-actin and -actinin, as indicated in the Materials and Methods. Boxed areas have been enlarged to highlight the filamentous and focal adhesion localization of -actinin. Identical results were also observed with oligo 2 (not shown). Bar, 50 µm.