Fig. 2

TTC36 blocks the binding of PELI1 to HPD. Immunoblotting analyses were performed with the indicated antibodies a–e. a LO2 cells expressing a control shRNA or Peli1-shRNA (left); LO2 cells expressing a control shRNA or Peli1-shRNA were treated with 10 μg mL⁻¹ cycloheximide for 0, 6, 12, or 18 h (middle); quantification of HPD levels, respectively, relative to 0 h is shown (right). Data represent the means ± s.d. of three independent experiments. ***P < 0.001, based on the Student’s t-test. b Flag-PELI1 or Flag-RNF138 were expressed in LO2 cells. The quantification numbers represent relative intensity of presented bands. c, e HEK293T cells were transfected with the indicated plasmids and MG-132 (10 μM) was added to the cells 12 h before they were harvested with a guanidine–HCl-containing buffer. A Ni-NTA pull-down assay was performed. d LO2 stable cells expressing Ttc36 shRNA#1 was stably transfected with a vector expressing RNAi-resistant (r) TTC36. Immunoprecipitation analyses with an anti-PELI1 antibody were performed