FIG. 3.

WSSV222 interacts with and ubiquitinates shrimp TSL in vitro. (A) A His₆-WSSV222 pull-down assay was performed using soluble extracts of E. coli or SF9-expressed WSSV222 mixed with GST-TSL. Soluble protein complexes were bound to GST beads and washed under high stringency condition before SDS-PAGE and immunoblot detection of His₆-WSSV222 and 222RING. Immunoblots were performed with anti-His₆ or anti-GST. (B) In vitro polyubiquitination assay performed using WSSV222 and 222RING with TSL in the presence of E1, E2, and ubiquitin. SDS-PAGE-resolved complexes were immunoblotted with anti-GST antibody. The negative control reaction was performed in the absence of E1 or E2 or with GST instead.