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Fig. 6. Effect of heterologous VILIP-1 and VILIP-3 expression on the subcellular localization of GC-B protein in hippocampal neurons with and without CNP-stimulation. Hippocampal cell cultures (24 hours after transfection) were analyzed by confocal immunofluorescence microscopy using affinity-purified polyclonal antibodies against GC-B and secondary Cy3 anti-rabbit antibodies (red). GFP, VILIP-1-GFP and VILIP-3-GFP expression (green) was detected as green fluorescence of the GFP-fusion protein following transient transfection of the vector constructs. Subcellular localization of GC-B (red) was detected with (A,C,E) or without CNP-stimulation (B,D,F) in GFP-transfected (A,B), VILIP-1-GFP-transfected (C,D) and VILIP-3-GFP-transfected (E,F) hippocampal neurons. Upper insets show co-localization of GC-B with VILIP-1-GFP, VILIP-3-GFP or GFP expression (A-F, green). The lower insets in A-F show the quantification of the pixel values (relative GC-B fluorescence intensity) from a cross section of the shown neurons as indicated with the yellow bars. Black bars in the insets indicate the localization of the cell surface membrane. Bar in A=20 µm.

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