Effect of nanobodies on APC-mediated histone H3 cleavage. APC (50 nM) and anti-APC nanobody (500 nM) were preincubated for 30 minutes. After the incubation, the mixtures were added to histone H3 (100 μg/mL). Over a period of 2 hours, samples were taken at different time points and added to reducing sample buffer for SDS-PAGEs. The 20 kDa molecular marker was used as a reference for normalization between gels. (A) Time course of H3 cleavage by APC in the absence nanobody (representative of 3 experiments). (B) Nine APC-specific nanobodies have minimal effects on APC-mediated H3 cleavage. The percent of H3 cleavage was calculated by normalizing with the H3 band intensity at time = 0 minute: 100% (intensity_{t=x min}/intensity_t=0 minute). The 20 kDa molecular weight (MW) marker was used as reference for normalization between gels. (C) SDS-PAGE of the enhancement of H3 cleavage by LP14. (D) Twelve nanobodies inhibited APC-mediated H3 cleavage. (E) SDS-PAGE of the potent inhibition of H3 cleavage by LP21. SDS-PAGE, sodium dodecyl sulfate polyacrylamide gel electrophoresis.