Fig. 3. Subcellular distribution of clathrin in non-transfected and VILIP-1-transfected C6 cells following CNP-stimulation as a measurement for membrane transport of GC receptor. (A) Western blot analysis using clathrin antibodies was performed with (+CNP) or without (–CNP) previous CNP-stimulation of non-transfected (C6) and VILIP-1-transfected (C6-V1) C6 cells and after separation of the P2 membrane fraction from the cytosolic fraction. (B) Quantification of the subcellular distribution of clathrin in non-transfected and VILIP-1-transfected C6 cells following CNP-stimulation. In non-transfected (C6) and VILIP-1-transfected (C6-V1) cells the association of clathrin with the P2 membrane fraction following CNP-stimulation was quantified using western blot analysis as shown in A. Mean values±s.e.m. are from seven experiments in B. Asterisks (*) mark a significant difference (P<0.05, n=7, Student's t-test). (C-F) Effect of GFP or VILIP-1-GFP expression on the subcellular localization of the transferrin receptor. C6 cells were transfected with GFP (E,F) or VILIP-1-GFP (C,D) and incubated with Rhodamine-transferrin for 13 minutes. After fixation the subcellular localization of Rhodamine-labelled transferrin (C-F, red channel) was observed by fluorescence microscopy and compared with VILIP-1-GFP (D, compare red and green channel) and GFP-expression (F, compare red and green channel). Bar in C=40 µm.