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. 2011 May 6;52(6):1068–1082. doi: 10.1093/pcp/pcr058

Fig. 1.

Fig. 1

Gene structure of ISA3 and substrate specificity of ISA3. (A) Gene structure of ISA3 on chromosome 9. The transposon Tos17 is inserted at the 3′ splice site of the 21st intron in the isa3 mutant line NC0371. Exons are indicated by boxes and the unspliced 11th intron in the full-length cDNA (AK101554) is indicated by a red box. (B) ISA3 protein levels in seed extracts of wild-type control (WT) and transformants expressing HA epitope-tagged ISA3 without the 11th intron (ISA3-HA) and with the intron [ISA3-HA (intron)] were compared by immunoblotting with anti-ISA3 and anti-HA epitope antibodies. (C) Zymogram analysis with recombinant ISA3 (rISA3) expressed in E. coli with different α-glucans. Vector control (−); HA epitope-tagged rISA3 (+). An equal amount (2 μl) of samples was loaded on native polyacrylamide gels for electrophoresis and then transferred to gels containing the indicated substrates. (D) Zymogram analysis with leaf and seed extracts. Crude extracts of the leaf (10 μl) and developing seed (5 μl) of wild-type Nipponbare control (WT) and isa3 were separated on a native polyacrylamide gel. Proteins were transferred to a PVDF membrane for immunoblotting with anti-ISA3 antibody (left panel) or a polyacrylamide gel containing β-limit dextrin for zymogram analysis (right panel). Cell extracts (2 μl) of E. coli transformed with a control vector (lane 1) and the vector containing HA epitope-tagged ISA3 (lane 2) were used as controls. The E. coli cell extracts for the immunoblotting analysis were diluted 5,000-fold relative to those used for the zymogram analysis. Note that rISA3 containing the HA epitope and His-tag at the C-terminus migrated more slowly than endogenous ISA3 bands extracted from the leaf and seed. Although specific fragments are recognized by the anti-ISA3 antibody (left panel), debranching activity of the endogenous ISA3 at the corresponding positions is under the limit of detection by zymogram analysis (right panel). Arrowheads indicate unidentified enzymes or complexes that modify the substrate in the gel but do not contain ISA3. Asterisks show non-specific fragments recognized by the ISA3 antibody.