Figure 4.

StauC forms a protein complex with Dcr-2a in cells. (A) Schematic representation for the recombinant StauC-S and Dcr-2a-V5-8xHis. (B) Sf9 cells were co-transfected with the gene expression constructs encoding L. decemlineata StauC-S and Dcr-2a-V5-8xHis. The transfected cells were incubated with or without dsGFP for 4 h before harvesting them at 48 h post-transfection. The proteins were etracted and used to perform western blot analysis using S and V5 tag-specific antibodies. 50 µg of total cell lysate was utilized to measure the levels of target proteins. (C) The same cells but exposed to DSP, a cell-permeable and reversible crosslinking agent, were analysed by western blot against S and V5 tags. 50 µg of total cell lysate was used for analysis. (D) The His-tagged protein and protein complexes extracted from the DSP-treated cells were subjected to immunoblotting using S and V5 tag-specific antibodies. (E) L. decemlineata StauC-S was expressed in Sf9 cells in the presence or absence of Dcr-2a. The protein complexes isolated from the DSP-treated cells by immortalized metal affinity chromatography were subjected to SDS-PAGE and western blot analysis