Figure 8.

Chaperoning activity of SBT4. 13PP and SPI-1. A SBT4.13 propeptide deletion mutant (SBT4.13ΔPP) was expressed in N. benthamiana leaves with or without co-expression of the SBT4.13 propeptide (SBT4.13PP) or SPI-1. Twenty micrograms of total leaf extract or 4 μg of apoplastic proteins were separated by 15% SDS-PAGE. Proteins were visualized by Coomassie staining and Western blotting as indicated. SBT4.13 and SBT4.13ΔPP (PP) were detected with a monoclonal mouse anti-His₆ antibody (1:5000; Dianova) in combination with a peroxidase-conjugated secondary antibody (1:10,000; Calbiochem) with enhanced chemiluminescence detection in apoplastic washes and total leaf extracts. Expression of SPI-1 was confirmed in total leaf extracts using a monoclonal FLAG M2-peroxidase conjugate (1:5000; Sigma-Aldrich). The molecular masses of marker proteins and that of SBT4.13 (black arrow) in the Coomassie-stained gel are indicated.