Fig. 1: The assembly and enzymatic activity of the ISA1-ISA2 complex.

a Gel filtration analysis of OsISA1, OsISA2 and OsISA1-ISA2 complex. Left, gel filtration chromatography. Right, SDS-PAGE corresponding to the chromatography. The star symbol indicates the contaminate Hsp70. b Analytical ultracentrifugation characterization of protein oligomerizations. The peak corresponded molecular masses are shown. c Enzymatic examination of OsISA1, OsISA2, and OsISA1-ISA2 complex using the BCA method. 4 nM OsISA1, 4 nM OsISA2, and 1 nM OsISA1-ISA2 was used in each reaction, respectively. The vertical ordinate shows the enzymatic activity in a unit of mg/s/μmol. mg, the amount of the reducing ends in the reactions; s, seconds of the reaction time; μmol, the enzymes in the reactions. At least three biological replicates were performed (n = 9; *p < 0.05; **p < 0.01; ***p < 0.001; unpaired t-test, error bars = mean ± SEM). d Native PAGE coupled enzymatic examination of OsISA1, OsISA2, and OsISA1-ISA2 complex. 50 nM protein was used for each lane. The native gel, containing 0.1% (w/v) maize amylopectin, is stained brown with I₂-KI solution. The debranching activity of isoamylase is indicated by white bands. Blank and BSA lanes were set as negative controls.