Journal: Journal of Biological Chemistry

Article Title: Neuronal Differentiation of P19 Embryonal Carcinoma Cells Modulates Kinin B2 Receptor Gene Expression and Function

doi: 10.1074/jbc.m502513200

Figure Lengend Snippet: FIG. 2. Detection of B2BKR expression in P19 cells during neuronal differentiation. A, Western blot. Forty g each of par- tially purified membranes of embryonal, undifferentiated P19 cells on day 0 and EBs on day 2 following treatment with RA were separated by SDS-PAGE. The proteins were transferred to a nitrocellulose membrane. The membrane was incubated with a polyclonal goat anti-B2BKR antiserum. The reaction was developed using the ECL reaction. Both undifferentiated cells and EBs express the B2BKR protein (45 kDa). B, detection of differential expression of B2BKRs by immunofluorescence. Following fixation, cells were incubated with an anti-polyclonal B2BKR antibody: undifferentiated P19 cells (UN, left panel), P19 cells at day 7 (middle panel), and day 9 (right panel) following induction to neuronal differentiation. A fluorescein isothio- cyanate-conjugated secondary antibody was used to detect the ex- pression of B2BKR. Counterstaining of cell nuclei was performed using DAPI. The expression of B2BKRs in undifferentiated cells was below the detection limit. Increasing percentages of cells expressing B2BKRs were detected on day 7 and 9 of neuronal differentiation. Scale bars indicate a size of 40 m. C, detection of co-expression of B2BKR and NF-200. For double-immunofluorescence staining of P19 cells on day 8 of differentiation, incubation of fixed cells with primary antibodies against the B2BKR and NF-200 was performed as detailed above, using Cy5-labeled (Zymed Laboratories Inc.) and Alexa-Fluo 488 (Molecular Probes)-labeled secondary antibodies, respectively, for detection. Fluorescence emissions were separated by using a confocal microscope. The left and middle panels show the images collected for B2BKR and NF-200 expression in the same cells. In the right panel, images are overlaid, indicating that B2BKRs are ex- pressed by neuronal differentiated cells.

Article Snippet: The proteins separated on the gel were electrophoretically transferred at 400 mA for 80 min onto a nitrocellulose membrane (Transblot, 0.45 m, Bio-Rad).

Techniques: Expressing, Western Blot, Purification, SDS Page, Membrane, Incubation, Quantitative Proteomics, Immunofluorescence, Double Immunofluorescence Staining, Labeling, Fluorescence, Microscopy