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Fig. 2. RNAi-mediated conditional knockdown of Dd-TRAP1. (A) Two PCR products (756 bp and 1198 bp) were ligated tail to tail, and inserted into the MB38 vector. The 756 bp inverted repeat (dark bar) and the 442 bp linker sequence (white bar) form a stem-loop RNA (dsRNA). (B) Growth kinetics of TRAP1-RNAi cells and parental MB35 cells. TRAP1-RNAi cells were cultured with 20 µg/ml of tetracycline () or without (), and cell-numbers were counted every 24 hours. As a control, the parental MB35 cells were also cultured without tetracycline (). At the exponential growth phase, when cell density reached over 5x10⁶ cells/ml, cells were diluted with fresh growth medium to a density of 1x10⁵ cells/ml. Total proteins were extracted every 24 hours for 7 days from these cultures, and western analyses were performed using the anti-Dd-TRAP1, anti-Dd-GRP94 and anti-Hsc90 antibodies.

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