Figure 4

A nucleotide-linked protein comigrates with pFOXC3-reverse transcriptase (RT) and is unaffected by strong base treatment. Products of reverse transcription reactions using mitochondrial ribonucleoprotein (mtRNP) particles isolated from a pFOXC3-containing strain or a plasmid-free strain (P-F) having [α-³²P]dATP and unlabeled deoxyguanosine triphosphate (dGTP) and thymidine triphosphate (TTP) were separated via 10% SDS-PAGE and transferred to nitrocellulose. (a) The two panels on the left are from a nitrocellulose membrane probed with protein A-purified pFOXC3-RT_55-68 rabbit antiserum and visualized by chemiluminesence. The panel on the right is a phosphorimage of radiolabeled protein on the same nitrocellulose membrane. Prestained protein size markers are indicated on the left in kDa. The gray arrow indicates non-specific bands detected in the plasmid-free mtRNP preparation. (b) Micrococcal nuclease (MN)-treated pFOXC3-containing mtRNPs were incubated with [α-³²P]dATP and unlabeled dGTP and TTP. The products were separated via 4-20% gradient SDS-PAGE and radiolabeled products were detected by a phosphorimager. The gel was rehydrated in 1 M KOH and, following incubation at 55°C, the gel was neutralized and dried prior to detection by a phosphorimager.