Figure 8.

BPLF1 inhibits the clearance of HTTQ103 aggregates. HeLa HTTQ103-CFP cells were transfected with BPLF1, BPLF1[C61A] or empty vector and treated with doxycycline for indicated times to stop de novo synthesis of HTT and the clearance of HTTQ103-CFP aggregates was monitored by filter trap assay. The clearance of HTTQ103-CFP aggregates is inhibited in the presence of catalytically active BPLF1. The same lysates were fractionated by SDS-PAGE and immunoblotted to control for BPLF1 expression and loading. (A) Representative western blots illustrating the persistence of HTTQ103-CFP aggregates in cells expressing BPLF1. (B) The clearance of aggregates was calculated as the difference between the intensity of the dots at time 0 and the intensity after blocking de-novo synthesis by treatment with doxycycline for 24 h or 48 h. Expression of catalytically active BPLF1 resulted in significantly reduced aggregate clearance after 24 h while more variable effects were observed after 48 h due to toxicity of the HTTQ103-CFP aggregates. Means ± SEM of three independent experiments. Statistical analysis was performed using Student t-test. *P ≤ 0.05