Figure 5

The C-terminal end of CaNDR1a is removed from the mature protein in tobacco. (a) Constructs used for transiently expressing HA- and HA-His-tagged CaNDR1a proteins in tobacco leaves. (b) Detection of CaNDR1a-tagged proteins by immunoblotting. The upper and lower panels show scanned films corresponding to membranes blotted with anti-HA and anti-His sera, respectively. For comparison, the same protein extracts were resolved by SDS-PAGE and subsequently transferred onto both membranes. Ten micrograms of proteins were loaded in each lane. Samples contained the main insoluble proteins that were extracted using SDS as described in the 'Methods' section. Lanes 1 & 2, negative controls (samples prepared from leaves expressing a GUS protein and non-infiltrated leaves, respectively); lane 3, HA-positive control, His-negative control (sample prepared from tissues expressing the N-terminally HA-tagged CaNDR1a protein); lanes 4-6, samples prepared from tissues expressing the doubly-tagged CaNDR1a protein (3 independent experiments); and lane 7, HA-negative control, His-positive control (sample prepared from Arabidopsis leaves constitutively expressing the C-terminally His-tagged AtBI1 protein) [56].