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. 2013 Mar 7;8(3):e50121. doi: 10.1371/journal.pone.0050121

Figure 2. Characterization of inducible PC12 cells stably expressing rCTFs.

Figure 2

Only the cell lines expressing rCTF-Q28-NES, rCTF-Q28-NLS and rCTF-Q13-NES are shown here (Other cells are shown upon request). (A) A timeline of rCTF expression in PC12 cells stably expressing rCTF-Q28-NES by quantitative real-time PCR (qRT-PCR). The rCTF mRNA level starts to be detected inDay3 and gradually increases with a time-dependent manner. The beta-actin expression level was used as an internal control. (B) A timeline of rCTF expression in PC12 cells stably expressing rCTF-Q28-NES by Western blot using the A6RPT-#5803 antibody. Protein expression starts to be detected on the fourth day after the Dox removal (“Day4”) and reaches abundant level in Day6. Anti beta-tubulin antibody was used as internal control. (C) Immunofluorescence cytochemistry in Day6 stable PC12 cell lines expressing rCTF-Q28-NES (upper row) and rCTF-Q28-NLS (lower row). The NES-tag faithfully anchored rCTF to the cytoplasm, whereas the NLS-tag efficiently directed rCTF to the nucleus. (D) Western blot analysis on protein extracts from stable cell lines expressing rCTF-Q28-NLS and rCTF-Q28-NES confirming efficient intracellular localizations (A6RPT-#5803: anti-Cav2.1CTF, beta-tubulin: a cytoplasmic protein marker, Histone H3: a nuclear protein marker). (E) Immunofluorescence cytochemistry in Day6 stable PC12 cell lines expressing rCTF-Q28-NES (upper row) and rCTF-Q13-NES (lower row). In PC12 cells expressing rCTF-Q28-NES, cytoplasmic aggregates are detected by both A6RPT-#5803 and 1C2. In rCTF-Q13-NES cells, rCTF-aggregates are recognized by the anti-Cav2.1 antibody A6RPT-#5803, but not by 1C2, a monoclonal antibody specific for expanded polyQ. (For C&E, scale bars: 10 µm).