Journal: PLoS ONE

Article Title: Channel Properties of Na x Expressed in Neurons

doi: 10.1371/journal.pone.0126109

Figure Lengend Snippet: ( A ) A RT-PCR analysis of the expression of Na x (upper) and glyceraldehyde 3-phosphate dehydrogenase (GAPDH, lower). Lane 1, parental Neuro-2a (N2a) cells cultured in DMEM containing 10% FCS; lanes 2–4, Neuro-2a cells cultured in serum-free medium with vehicle (lane 2), 1 mM dbcAMP (lane 3), or 20 μM retinoic acid (RA) (lane 4); lanes 5 and 6, a stable transfectant of Neuro-2a cells with pTRE-FLAG-mNa x (N2a-Mf1) maintained under serum-free conditions (lane 5) and N2a-Mf1 cells infected with the Tet-off adenovirus for the expression of FLAG-mNa x (lane 6). The parental N2a cells were Na x -negative. GAPDH was amplified from the same cDNA preparation as Na x . ( B ) Western blotting with anti-mNa x . Cells were cultured as in A . The lower panel shows Coomassie Brilliant Blue (CBB) staining to verify the amount of protein applied. ( C and D ) Anti-Na x immunocytochemistry of cells cultured as in A using a wide-field fluorescence microscope ( C ) and confocal laser scanning microscope ( D ). PhC, phase contrast; DIC, differential interference contrast image. Scale bars, 20 μm for ( C ) and 10 μm for ( D ). ( E ) Na + imaging to confirm the functional expression of FLAG-tagged Na x in N2a-Mf1 cells. C6M16 cells expressing non-tagged mouse Na x and C6Mf4 cells expressing FLAG-tagged Na x were analyzed to determine whether the FLAG tag affected the gating of Na x channels. C6M16, C6Mf4, and N2a-Mf1 cells showed similar [Na + ] o -sensitive responses. Their parental N2a and C6 cells, which were Na x -negative, did not show [Na + ] o -sensitive responses. The ordinate shows the change observed in the fluorescence ratio (ΔF, 340/380 nm). The fluorescence ratio at 0 min was set as the zero point on the ordinate. The extracellular perfusion solution was changed from the 145 mM Na + solution to the 170 mM Na + solution at 0 min. Data represent the mean ± SE (n = 25 for each). Uncropped images of gels and blots are shown in .

Article Snippet: Fluorescence was observed with a wide-field fluorescence microscope (BZ8000, Keyence) or laser scanning confocal microscope (A1R, Nikon).

Techniques: Reverse Transcription Polymerase Chain Reaction, Expressing, Cell Culture, Transfection, Infection, Amplification, Western Blot, Staining, Immunocytochemistry, Fluorescence, Microscopy, Laser-Scanning Microscopy, Imaging, Functional Assay, FLAG-tag