Figure 5abc.

Amyloidogenic M-TTRs are degraded by proteasome. (A–E) HeLa cells were transfected with the indicated TTR constructs. After 42 h, cells were treated with 500 μM cycloheximide and the proteasome inhibitor, MG132 (30 μM) or DMSO. Cell lysate and media were collected immediately (0 h) or 2, 6 h after the addition of cycloheximide. Densitometric quantifications were performed on the intracellular TTR and secreted TTR from HeLa cells. Intracellular TTR expression at different time points was calculated as relative expression: relative expression (%)=100 × (intracellular TTR at given time t/intracellular TTR at 0 h). Secreted TTR was calculated as follows: (secretion level at given time t/secretion level at 0 h). White circles, CHX and black circles, CHX+MG132. (F) The % relative expression level of intracellular M-TTRs at 6 h after cycloheximide treatment in HeLa cells untreated or treated with 30 μM MG132. (G) The secretion ratio of wild-type or non-amyloidogenic M-TTRs at 6 h after cycloheximide treatment in HeLa cells untreated or treated with 30 μM MG132. Conditions in (F) and (G) are identical to those described above. All experiments were performed in triplicate. Values represent the mean±s.e. (^*P<0.05, ^**P<0.01, ^***P<0.001).