Retroviral vector-mediated expression of mJagged2 in the Rab-9 cells.

Rab-9 cells were infected with retroviral vectors LXSN (empty vector control), LMJSN (expressing full-length mJagged2), and LECDSN (expressing the extracellular domain of mJagged2) and selected with G418. (A) Northern blot analysis using 5 μg total RNA in each lane and probed with either the full-length mJagged2 probe (MJS; upper panel) or the intracellular domain probe (ICD; lower panel). The position of the 28S rRNA is shown. (B) Western blot analysis using antisera against the intracellular domain of mJagged2. Ten micrograms total cell lysate from each cell line was used in each lane. EML C1 served as the positive and size (150-kd) controls, whereas MPRO served as the negative control. Rab-9/LMJSN expressed mJagged2 of correct size. No specific bands were detected when the blot was probed with preimmune serum (not shown). (C-E) Indirect immunofluorescence staining using rabbit anti-ICD antibodies against the intracellular domain of mJagged2, followed by fluorescein isothiocyanate-conjugated donkey antirabbit IgG (green fluorescence). Cells were double stained with propidium iodide (red fluorescence) to reveal the nuclei. (C) Rab-9/LXSN. (D) Rab-9/LMJSN. (E) Rab-9/LECDSN. Only Rab-9/LMJSN showed positive staining with clonal variation in the level of expression. Staining with preimmune or anti-GST sera was negative (not shown).