Figure 6. Repair of gapped and gapped 5′-THF substrates in brain and mucosa is abolished by pol β neutralizing antibody.

Reactions with gapped (A, lanes 1–7) and gapped-5′-THF (B, lanes 8–18) substrates were assembled with brain and mucosal extracts or with purified pol β. Duplicate reactions were assembled w/o preincubation with the pol β neutralizing antibody. In all cases the antibody blocked gap filling synthesis. Inhibition was proportional to antibody amount: lane 10 shows a significant reduction of the [+1] product with 1 μl, while lane 12 shows a lesser reduction using 0.5 μl antiserum. With nuclear extracts a similar amount of antibody was sufficient to block gap filling synthesis and subsequent ligation (lanes 14, 16, 18). In the case of mucosa, both products (lane 17) were seen following 8- and 15-minute reactions. B) For the gap-5′-THF assays (lanes 8–18) substrates (bottom) and products (top) are shown at different exposures to clearly visualize elimination of the [+1] synthesis product by pol β neutralizing antibody. C) Western blotting analysis shows similar levels of pol β protein in nuclear extracts from the brain and mucosa, while PCNA is detected only in mucosal extracts. Ligase lll and FEN1 are detected in both extracts; higher levels of FEN1 were observed in mucosa compared to the brain. Reprobing for actin served as a loading control.

Immunoreactivity levels are depicted in bar graphs representing densitometric measurements of signals for brain relative to mucosal extracts and expressed as means ± SEM (n=4). ND-not detected.