Fig. 7. SCA14 mutant PKC reduces ERK2 nuclear accumulation and phosphorylation. (A) Confocal images of HeLa cells transiently transfected with GFP-ERK2 and cotransfected with either wild-type PKC-RFP or PKC G118D-RFP (V138E and C142S mutants confocal data not shown). Activation of PKC with PMA induced GFP-ERK2 nuclear accumulation in time. Images shown are recorded 250 and 500 seconds after addition of 400 nM PMA. Scale bar: 10 µm. (B) Translocation analysis shows that GFP-ERK2 nuclear translocation was reduced in cells coexpressing SCA14 mutant PKC when compared with cells that express only GFP-ERK2 or coexpress wild-type PKC-RFP. (C) Bars represent the ratio of nuclear/cytosolic ERK fluorescence in the absence and presence of wild-type PKC and SCA14 mutant PKC (unpaired t-test, ^**P<0.01; ^***P<0.001). (D) Western blot analysis showed that endogenous ERK1 and ERK2 are phosphorylated after PKC activation with 400 nM PMA for 10 minutes. Reduced ERK2 phosphorylation was observed in the presence of SCA14 mutant PKC (G118D, V138E and C142S) compared with cells expressing wild-type PKC. Furthermore, equal ERK and PKC levels were observed using anti-ERK and anti-PKC antibodies. (E) Quantification of the band intensity. Bar graph represents the phosphorylation of ERK normalized to the amount of ERK and PKC and related to the value obtained for cells expressing wild type PKC. Data in C and E represent mean ± s.e.m.