FIG. 5.
K137R-AAV8 lysine mutant vector demonstrates reduced ubiquitination in comparison to WT-AAV8 vector. (A) Approximately 3×108 viral particles of WT-AAV8 and K137R-AAV8 vectors were denatured at 95°C for 5 minutes. The denatured viral particles were then used to perform the ubiquitin conjugation assay according to the manufacturer's protocol. The processed samples were electrophoresed on a 4–20% denaturing polyacrylamide gel and the ubiquitination pattern detected by immunoblotting using an anti-ubiquitin antibody. The mono-to-polyubiquitin conjugates are detected as a smear at molecular weight >150Kda. (B) Approximately 3×108 viral particles of WT-AAV8 and K137R-AAV8 vectors were denatured with radioimmunoprecipitation assay (RIPA) buffer containing protease inhibitor cocktail at 95°C for 10 minutes. The samples were resolved in a 4–20% denaturing polyacrylamide gel and the VP1 (87Kda), VP2 (72 KDa), and VP3 (62 Kda) capsid proteins were detected with AAV clone B1 antibody as described in the Materials and Methods section.