Fig. 3. Specificity of anti-DIP13 antiserum and DIP13 expression during the cell cycle. (A) Characterization of a polyclonal anti-DIP13 antiserum produced in rabbit. (Left panel) Crude cell extracts from 10⁶ C. reinhardtii cells per lane were probed with preimmune serum (P; 1:500) as well as crude (C, 1:500) and affinity-purified (A, 1:100) anti-DIP13 antiserum and detected by enhanced chemiluminescence. Sizes of reacting bands are indicated to the left. (Right panel) Crude cell extracts of 10⁶ C. reinhardtii cells per lane were probed with affinity-purified anti-DIP13 antibody preincubated with 80 µg recombinant DIP13 per ml incubation buffer (A-PI) or affinity-purified anti-DIP13 antibody (A, 1:100). The size of the reacting band is indicated to the right. (B) The 24 hour C. reinhardtii life cycle under a synchronizing 16 hour light/8 hour dark regimen. Every full hour 100 cells from a synchronous culture were analyzed by light microscopy and absolute numbers of cells in the 1-, 2-, 4- or 8-cell stage were noted in the table shown on the right. (C) Western analysis with samples isolated from the same culture as in B during the period of cell division. Equal amounts of protein per lane were probed with affinity-purified anti-DIP13 antibody (DIP13), anti--tubulin antibody (Tub) and anti-L23 antibody (L23). Time points of protein sampling correlate with time points in B. The same membrane was incubated successively with the three primary antibodies. Exposure times to film were 1 minute in each case. (Lower panel) Coomassie-stained gel of identical protein samples as a loading control. St, standard lane.