Fig. 1. Characterisation of a Cdc25B peptide antibody. (A) The guinea pig anti-Cdc25B antibody B1:5 recognises YFP-Cdc25B, but not YFP-Cdc25A. Cell lysates from HeLa cells transfected with YFP-Cdc25A (lane A) and YFP-Cdc25B (lane B) were subjected to immunoblotting using B1:5 (anti-Cdc25B) and anti-GFP antibodies as indicated in the figure. (B) The B1:5 antibody recognises Cdc25B in whole cell lysates. HeLa cells were transfected with siRNA against Cdc25A (lane A) or Cdc25B (lane B). Cell lysates were harvested 48 hours after transfection and analysed by immunoblotting using the B1:5 antibody. (C) Assessment of antibody specificity by RNA interference. HeLa cells were microinjected with siRNA against Cdc25B (upper panel) or lamin A/C (lower panel) together with a pCFP-Golgi plasmid to mark injected cells. Thirty-five hours later, cells were fixed and stained with B1:5 and cyclin B1 antibodies. The B1:5 antibody stained all cells expressing cyclin B1. In cells microinjected with Cdc25B siRNA, but not with lamin A/C siRNA, this staining is diminished. Cells expressing cyclin B1 that also are microinjected with siRNA are indicated by the letter a.