Fig. 2. SCA14 mutations located in C1B affect C1B translocation kinetics upon phorbol ester stimulation. (A) Confocal images of HeLa cells cotransfected with C1B-GFP and SCA14 mutant C1B-RFP. PMA activation induces irreversible translocation of C1B-GFP and C1B G118D-RFP to the plasma membrane. By contrast, the V138E and C142S C1B-RFP did not respond to PMA activation. Images shown were recorded prior to (0) and 200 seconds after addition of 400 nM PMA. Scale bars: 10 µm. (B) Translocation analysis of cotransfected C1B-GFP and mutant G118D, V138E or C142S C1B-RFP showed that the V138E and C142S mutations impair phorbol ester (400 nM PMA)-induced membrane translocation. Each of the traces in B and C represent mean ± s.e.m. of 4-6 cells from at least three independent experiments.