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. 2007 Jan;143(1):364–377. doi: 10.1104/pp.106.090050

Figure 6.

Figure 6.

rEPIC2B inhibits apoplastic tomato proteases including PIP1. A, EPIC2B inhibits apoplastic Cys proteases of tomato. Intercellular fluids of tomato were preincubated with E-64 (1.02 μm) and combinations of EPIC2B (2.00 μm) and EPI1 (2.00 μm) for 1 h before DCG-04 was added to the final concentration of 0.22 μm to label Cys proteases with biotin. Following labeling, proteins were acetone precipitated, and biotin-labeled proteases were captured with magnetic streptavidin beads and analyzed by SDS-PAGE and streptavidin western blotting. The compositions of the reaction mixes are indicated by the ± signs at the top. Molecular mass markers are shown on the left. Biotin-labeled proteases are indicated by dots on the right. Black dot, gray dot, and white dot indicate biotin-labeled protease bands of 30 kD, 36 kD, and 40 kD, respectively. B, PIP1 has Cys protease activity. Intercellular fluids of N. benthamiana expressing PIP1-His were first incubated with 0.22 μm DCG-04 for 2 h. Proteins were acetone precipitated, and biotin-labeled proteases were captured either by magnetic streptavidin beads (lane 1) or by magnetic Ni-NTA beads to purify His-tagged proteins (lane 2). Extracted proteins were separated by SDS-PAGE and subjected to western blotting with streptavidin. The molecular masses of the markers in lane M are shown on the left. The band corresponding to the DCG-04-labeled PIP1-His is indicated with an arrow. C, EPIC2B is an inhibitor of PIP1. Time-course labeling of PIP1-His with DCG-04 was performed in the absence or presence of rEPIC2B. Intercellular fluids of N. benthamiana expressing PIP1-His were preincubated with rEPIC2B (2,240 nm) or the irreversible protease inhibitor E-64 (1,120 nm) prior to labeling with DCG-04 (220 nm). The labeling reactions were stopped at five time points (10, 20, 30, 60, and 120 min, respectively) by adding ice-cold acetone. PIP1-His was recovered from precipitated proteins by magnetic Ni-NTA beads and analyzed by SDS-PAGE and streptavidin western blotting. Note that rEPIC2B competitively inhibits binding of DCG-04 to PIP1 at the 30-min labeling time point but not later, suggesting that it is a reversible inhibitor. Approximate molecular masses of the labeled PIP1-His proteins are shown on the left side.