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. 2013 Sep 23;8(9):e75188. doi: 10.1371/journal.pone.0075188

Figure 6. Knockdown of Hsp70 inhibits JEV subgenomic replicon replication.

Figure 6

293T cells was transfected with JEV replicon RNA at 24 h post-transfection with sh-Hsp70 or sh-NC. (A) Cells were harvested at 12, 24, 36 h post-transfection respectively. Total cellular RNA was extracted and subjected to RT reaction. The level of JEV RNA (NS5) was determined by real-time PCR. The results shown are from three independent assays, with the error bars representing the standard deviations. (B) Cell lysates collected at 36 h p.i. were subjected to immunoblotting with antibodies against JEV NS3, NS5, Hsp70 and GAPDH. The protein levels were quantified by immunoblot scanning and normalized with respect to the amount of GAPDH (right panel). Error bars represent the standard deviations of results from three independent assays (***, P < 0.0001, student’s t-test). (C) Cell lysates collected at 36 h p.i. were subjected to luciferase assay (**, P < 0.001, student’s t-test).