Western blots of crude plant extracts and of purified ZERA-16E7SH and ZERA-eGFP protein. (A) Samples from plants expressing ZERA-16E7, ZERA-16E7SH and 16E7SH were harvested at 3, 5, 7 and 10 dpi, separated on a polyacrylamide gel and blotted onto nitrocellulose membrane. Proteins were detected with HPV-16 E7 antibody. 0.6 μg of E. coli- produced purified His-E7 protein was used as a positive (+ve) and comparative control. SH indicates the use of the shuffled 16E7 gene. + or - indicates the fusion of Zera®. Black arrows indicate the E7 positive control protein (18 kDa) and the Zera®-fused E7 proteins. (B) Leaves from vacuum-infiltrated N. benthamiana co-infiltrated with pBIN-NSS and either pTRAc-ZERA-16E7SH or pTRAc-ZERA-eGFP were extracted 5 dpi, ground in liquid nitrogen, homogenized, filtered and separated on sucrose density gradients. Interphase fractions (IF) were aspirated, pellets (P) were resuspended and run on acrylamide gels, blotted on nitrocellulose membranes and probed with HPV-16 E7 monoclonal antibody or anti-GFP monoclonal antibody. Black arrows indicate protein bands of expected sizes.