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. 2013 Oct 5;42(1):396–416. doi: 10.1093/nar/gkt898

Figure 5.

Figure 5.

Velocity sucrose gradient fractionation of A3C-WT or mutant proteins and L1 ORF1p containing complexes. Whole-cell lysates from HeLa cells co-transfected with plasmids expressing WT or mutant A3C proteins and L1 reporter plasmid pJM101/L1RP were layered over 10–50% sucrose containing Mg2+. The lowest number fraction corresponds to the bottom of the gradient. ORF1p (40 kDa), A3C WT or mutant (24 kDa) and S6 ribosomal marker proteins (32 kDa) were detected by immunoblot analysis using anti-ORF1p, anti-HA and anti-S6 antibodies, respectively. In the absence of ectopically expressed A3C proteins (pcDNA 3.1) and in the presence of overexpressed W74A or R122A mutant proteins, ORF1p co-localizes predominantly with ribosomes and polyribosomes in the bottom portion of the gradient. Co-expression of ORF1p and A3C-WT or C97S/C100S mutant protein shifted ORF1p to fractions 5, 6 and 7 (13–19% sucrose) in which ORF1p and A3C-WT or C97S/C100S mutant containing complexes were detectable. Cell lysates from 2102EP cells expressing endogenous L1 ORF1p were loaded as positive control for ORF1p expression.