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Targeting of 2A constructs and 2A cleavage efficiency.

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posted on 14.12.2012, 01:29 by Stefan Burén, Cristina Ortega-Villasante, Krisztina Ötvös, Göran Samuelsson, László Bakó, Arsenio Villarejo

(A–H) Bright field and confocal images of Arabidopsis protoplasts transiently expressing CFP-2A-RABD2a (A and B), SP-CFP-2A-RABD2a (C and D), GS-CFP-2A-RABD2a (E and F) and CFPGUS-2A-RABD2a (G and H). Bars = 5 µm. (I) Immunoblot analysis of protein extracts from Arabidopsis protoplasts transiently transfected with non-tagged RABD2a (negative control), CFP (positive control), CFP-2A-RABD2a, SP-CFP-2A-RABD2a, GS-CFP-2A-RABD2a and CFPGUS-2A-RABD2a using anti-GFP antiserum. 2A cleaved (*), non-cleaved full-length 2A polyproteins (**) and putative degradation products (−) are indicated. Protein loading was adjusted in order to highlight both cleaved and non-cleaved product for each construct. (J) Cleavage efficiency of the different 2A constructs was estimated from the amounts of cleaved (*) versus non-cleaved (**) products as described in Material and Methods. Error bars show standard error (n = 4, *** = p<0.001).