Figure 8

Countin mRNA and protein levels in BS153 and the parental strain, Ax4. A) Total RNA was isolated from cells growing on bacteria (0) and at the times indicated (in hours) after the onset of development. RT-PCR was carried out using oligonucleotides specific for countin mRNA (upper band in each panel). H7 specific oligonucleotides were used as an internal control (lower band in each panel). B) Ax4 and BS153 cells were washed free of bacteria, resuspended in PDF buffer at 5 × 10⁶ cells/ml, and were incubated with shaking for the times indicated (in hours). For cellular Countin, samples were removed, and the cells were pelleted and washed. Total protein was isolated from the cells, and equal amounts of each protein sample were separated by PAGE, blotted, and probed with antiserum specific for countin (Cell panel). The conditioned medium (CM) from each sample, after removal of the cells, was used as a source of secreted Countin. The proteins from equal volumes of the CM samples were separated by PAGE, blotted, and probed with antiserum specific for Countin (CM panel). The 6 and 12 hour conditioned medium samples were concentrated, and the proteins from equivalent volumes were separated by PAGE, blotted, and probed with antiserum specific for conditioned medium factor (CMF panel).