Fig. 4. SCA14 mutant PKC shows increased membrane targeting but does not affect C2 domain functioning. (A) Translocation analysis of HeLa cells cotransfected with wild-type PKC-RFP and wild-type PKC-GFP. Both proteins induced rapid reversible translocation to the plasma membrane upon histamine stimulation. No difference was observed in translocation pattern between the two wild-type PKC proteins fused to different fluorophores. Furthermore, wild-type PKC-RFP was cotransfected with SCA14 mutant G118D (B), V138E (C) and C142S (D) PKC-GFP. No difference was observed in membrane dissociation between wild-type and SCA14-mutant PKC. Increased translocation amplitudes were observed for SCA14 mutant PKC compared with wild-type PKC. Graphs show the translocation curve of single cells, but represent three independent experiments. (E) Quantification of the amount of PKC translocated to the plasma membrane upon histamine stimulation. Bars represent the ratio between the maximal amplitude of wild-type PKC and the maximal amplitude of SCA14-mutant PKC. Data are means ± s.e.m. of three experiments each on three cells (unpaired t-test, ^**P<0.01; ^***P<0.001). (F) No differences were observed in C2 domain-induced translocation kinetics of full-length PKC-GFP and mutant G118D, V138E or C142S PKC-GFP in response to 5 µM A23187. The curves are mean ± s.e.m. of 3-5 cells and the data are representative of three experiments.