Figure 2.
(A) Western blot with a discoidin-specific antibody on protein isolated from wild-type AX2 cells (control) and from four of nine randomly chosen clones silenced by discoidin RNAi (top). mcRNAi1 and 2 display complete silencing, and mcRNAi 3 and 4 display partial silencing. A Northern blot with RNA from the same cell lines, hybridized with a discoidin-specific probe, is shown in the middle. As a control for equal loading, ethidium bromide-stained large rRNA is shown in the bottom. The RNAi construct was introduced into cells via a multicopy vector (mc). (B) Protein and RNA were isolated from an AX2 strain carrying the G418 resistance gene under control of the actin 6 promoter (A6P-NPT) and from the discoidin RNAi strain (mcRNAi2) expressing the RNAi from the same promoter grown in axenic medium to a density of 2 × 106 cells/ml (I), grown in bacterial suspension to a density of 3 × 106 cells/ml (II), grown in bacterial suspension to a density of 3 × 106 cells/ml and then developed in shaking culture for 5 h (III), and grown in axenic culture to a density of 2 × 106 cells/ml after passage through bacterial growth and development for 5 h (IV). First line, cellular protein separated by SDS-PAGE, blotted, and probed for discoidin. Second line, Northern blot probed for discoidin mRNA. Third line, slot blot probed for NPT mRNA. Fourth line, ethidium bromide-stained large rRNA as a loading control.